CN112119157A - Prostate specific membrane antigen CAR and methods of use thereof - Google Patents

Abstract

The present disclosure provides modified immune cells (e.g., modified T cells) comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) (e.g., human PSMA). The present disclosure provides modified immune cells (e.g., modified T cells) comprising a CAR having affinity for PSMA and a dominant negative receptor and/or switch receptor. The present disclosure provides modified immune cells (e.g., modified T cells) comprising a CAR having affinity for PSMA and a dominant negative receptor and/or switch receptor, wherein the modified cells are capable of expressing and secreting a bispecific antibody.

Description

Prostate specific membrane antigen CAR and methods of use thereof

Cross Reference to Related Applications

This application entitled priority to U.S. provisional application 62/639,321 filed on 3, 6, 2018, pursuant to 35 u.s.c. § 119(e), which is incorporated herein by reference in its entirety.

Background

The main challenge in the application of immunotherapy to solid malignancies is to break the tolerance to self-antigens. Vaccine strategies aimed at utilizing endogenous anti-tumor T cells are limited by the T Cell Receptor (TCR) repertoire, which can be deleted within the thymus as part of central tolerance or rendered non-functional by the post-thymic mechanisms of peripheral tolerance. One strategy to overcome such obstacles is to use the Chimeric Antigen Receptor (CAR) pathway to generate genetically engineered T cells that are redirected to tumor antigens. CAR T cells use lymphocytes from the patient programmed with a gene transduced with a chimeric receptor gene to combine the antigen recognition domain of a specific antibody with the signaling domain of the TCR.

Prostate Specific Membrane Antigen (PSMA) is a membrane-bound protein that is expressed on the surface of cells and is reported to be highly overexpressed in prostate cancer tissues. PSMA expression is directly linked to the progression of tumor grade and stage and is thought to confer a selective growth advantage on prostate cancer cells. Thus, PSMA may be an ideal target for immunotherapy against prostate cancer.

Another major challenge in cancer immunotherapy is the hostile microenvironment in which the targeted tumor is located. For example, immunosuppressive receptor ligands, such as PDL1(CD274) that binds to PD1(CD279), upregulate and negatively regulate T cell activity in the tumor microenvironment. Additionally, TGF-. beta.s overexpressed in prostate tumor cells may act as immunosuppressive molecules.

Thus, there is a need in the art for novel cancer immunotherapies targeting PSMA. The present invention satisfies this need.

Disclosure of Invention

The present invention is based on the following findings: human and murine Prostate Specific Membrane Antigen (PSMA) Chimeric Antigen Receptor (CAR) T cells exhibit potent anti-tumor activity. The invention is also based on the following findings: PSMA-CAR T cells including a dominant negative receptor (dominant negative receptor) and/or a switch receptor (switch receptor) exhibited significantly enhanced anti-tumor activity.

Thus, in certain aspects, the present disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain; and dominant negative receptors and/or switch receptors.

In certain exemplary embodiments, the PSMA-binding domain is a murine PSMA-binding domain.

In certain exemplary embodiments, the PSMA-binding domain is a human PSMA-binding domain.

In certain exemplary embodiments, the PSMA-binding domain is selected from an antibody, Fab, or scFv.

In certain exemplary embodiments, the scFv comprises the amino acid sequence set forth in any one of SEQ ID NOs 13, 14, 26, 38, 50, or 62.

In certain exemplary embodiments, the CAR comprises a transmembrane domain, and an intracellular domain.

In certain exemplary embodiments, the transmembrane domain comprises a transmembrane region derived from CD 8.

In certain exemplary embodiments, the CD 8-derived transmembrane region comprises the amino acid sequence set forth in SEQ ID NO: 88.

In certain exemplary embodiments, the transmembrane domain further comprises a hinge region derived from CD 8.

In certain exemplary embodiments, the hinge region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO 86.

In certain exemplary embodiments, the intracellular domain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain.

In certain exemplary embodiments, the intracellular domain comprises an ICOS signaling domain and a CD3 zeta signaling domain.

In certain exemplary embodiments, the intracellular domain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain.

In certain exemplary embodiments, the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 92.

In certain exemplary embodiments, the ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID NO 203.

In certain exemplary embodiments, the variant ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID No. 95.

In certain exemplary embodiments, the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO 97 or 100.

In certain exemplary embodiments, the dominant negative receptor is a truncated variant of the wild-type protein that is associated with a negative signal.

In certain exemplary embodiments, the truncated variant of the wild-type protein that is associated with a negative signal comprises the amino acid sequence set forth in SEQ ID NO: 115.

In certain exemplary embodiments, the switch receptor comprises a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal; and a second domain, wherein the second domain is derived from a second polypeptide that is associated with a positive signal.

In certain exemplary embodiments, the first domain comprises at least a portion of an extracellular domain of a first polypeptide that is associated with a negative signal, and wherein the second domain comprises at least a portion of an extracellular domain of a second polypeptide that is associated with a positive signal.

In certain exemplary embodiments, the switch receptor further comprises a switch receptor transmembrane domain.

In certain exemplary embodiments, the switch receptor transmembrane domain comprises a transmembrane domain of a first polypeptide associated with a negative signal; or a transmembrane domain of a second polypeptide associated with a positive signal.

In certain exemplary embodiments, the first polypeptide associated with a negative signal is selected from CTLA4, PD-1, BTLA, TIM-3, and TGF β R.

In certain exemplary embodiments, the second polypeptide associated with a positive signal is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD 1; a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD 28; and a second domain comprising at least a portion of the intracellular domain of CD 28.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO 117.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD 1; a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of PD 1; and a second domain comprising at least a portion of the intracellular domain of CD 28.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO: 119.

In certain exemplary embodiments, the first domain comprises at least a portion of the extracellular domain of PD1 that contains an alanine (a) to leucine (L) substitution at amino acid position 132.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO. 121.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1, which comprises a substitution of alanine (a) to leucine (L) at amino acid position 132, and a second domain comprising at least a portion of the intracellular domain of CD 28.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO. 121.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of PD1, which comprises a substitution of alanine (a) to leucine (L) at amino acid position 132, and a second domain comprising at least a portion of the intracellular domain of 4-1 BB.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO: 215.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of TIM-3; and a second domain comprising at least a portion of the intracellular domain of CD 28.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO: 127.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of a TGF β R; and a second domain comprising at least part of the intracellular domain of IL12R β 1.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO 123.

In certain exemplary embodiments, the switch receptor comprises a first domain comprising at least a portion of the extracellular domain of a TGF β R; and a second domain comprising at least part of the intracellular domain of IL12R β 2.

In certain exemplary embodiments, the switch receptor comprises the amino acid sequence set forth in SEQ ID NO 125.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO 213 or 215.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO 117 or 119.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO. 121.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 127.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO 123.

In another aspect, the disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 14, 16, 38, 50, or 62; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 125.

In another aspect, the present disclosure provides a modified immune cell or precursor cell thereof comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14; and a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

In certain exemplary embodiments, the modified cell secretes a bispecific antibody.

In certain exemplary embodiments, a bispecific antibody comprises a first antigen-binding domain and a second antigen-binding domain.

In certain exemplary embodiments, the first antigen binding domain binds to a negative signal selected from CTLA4, PD-1, BTLA, TIM-3, and TGF β R.

In certain exemplary embodiments, the second antigen-binding domain binds to a costimulatory molecule.

In certain exemplary embodiments, the co-stimulatory molecule is CD 28.

In certain exemplary embodiments, the modified cell is a modified T cell.

In certain exemplary embodiments, the modified T cell is an autologous cell.

In certain exemplary embodiments, the modified cell is a Cytotoxic T Lymphocyte (CTL).

In certain exemplary embodiments, the modified cell is a Natural Killer (NK) cell.

In certain exemplary embodiments, the modified cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

In certain exemplary embodiments, the modified cells are autologous cells.

In certain exemplary embodiments, the modified cell is derived from a human.

In certain exemplary embodiments, the modified T cell is derived from a human.

In another aspect, the disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain; and a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor.

In certain exemplary embodiments, the first nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 106, 108, 110, 112, 114, 210, 212.

In certain exemplary embodiments, the second nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 116, 118, 120, 122, 124, 126, 128, 214, or 216.

In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker.

In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding an Internal Ribosome Entry Site (IRES).

In certain exemplary embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide.

In certain exemplary embodiments, the self-cleaving peptide is a 2A peptide.

In certain exemplary embodiments, the 2A peptide is selected from the group consisting of porcine teschovirus-12A (P2A), sphingan nudus beta-tetrad virus (thoseaagigna virus)2A (T2A), equine rhinitis a virus (E2A), and foot-and-mouth disease virus (F2A).

In certain exemplary embodiments, the 2A peptide is T2A.

In certain exemplary embodiments, the 2A peptide is F2A.

In certain exemplary embodiments, an isolated nucleic acid comprises, from 5 'to 3', a first nucleic acid sequence, a linker, and a second nucleic acid sequence.

In certain exemplary embodiments, the isolated nucleic acid comprises, from 5 'to 3', a second nucleic acid sequence, a linker, and a first nucleic acid sequence.

In another aspect, the disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the nucleic acid sequence recited in any of SEQ ID NOs 180, 15, 27, 39, 51, or 63; and a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor comprising the nucleic acid sequence set forth in any one of SEQ ID NOs 116, 118, 120, 122, 124, 126, 128, 214, or 216.

In another aspect, the disclosure provides an isolated nucleic acid comprising a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA-binding domain comprising the nucleic acid sequence set forth in SEQ ID NO: 180; and a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor comprising the nucleic acid sequence set forth in SEQ ID NO. 116.

In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding T2A.

In certain exemplary embodiments, the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding F2A.

In another aspect, the disclosure provides an isolated nucleic acid comprising the nucleic acid sequence recited in any one of SEQ ID NOs 152-168, 210, 212, and 217-226.

In another aspect, the disclosure provides an isolated nucleic acid comprising the nucleic acid sequence encoding the bispecific antibody recited in any one of SEQ ID NOs 130, 132, 134, 136, or 138.

In another aspect, the present disclosure provides an expression construct comprising the isolated nucleic acid of any one of the above embodiments.

In certain exemplary embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

In certain exemplary embodiments, the expression construct is a lentiviral vector.

In certain exemplary embodiments, the lentiviral vector further comprises an EF-1 alpha promoter.

In certain exemplary embodiments, the lentiviral vector further comprises a Rev Response Element (RRE).

In certain exemplary embodiments, the lentiviral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In certain exemplary embodiments, the lentiviral vector further comprises a cPPT sequence.

In certain exemplary embodiments, the lentiviral vector further comprises an EF-1 alpha promoter, a Rev Response Element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

In certain exemplary embodiments, the lentiviral vector is a self-inactivating lentiviral vector.

In another aspect, the present disclosure provides a method of producing a modified immune cell of any one of the above embodiments or a precursor cell thereof, comprising introducing one or more of the nucleic acid of any one of the above embodiments or the expression construct of any one of the above embodiments into an immune cell.

In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cell of any one of the above embodiments.

In certain exemplary embodiments, the method further comprises administering lymphodepleting chemotherapy (lymphodepleting chemotherapy) to the subject.

In certain exemplary embodiments, the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and/or fludarabine (fludarabine).

In another aspect, the present disclosure provides a method of treating prostate cancer in a subject in need thereof. The method comprises administering to the subject a lymphodepleting chemotherapy comprising a therapeutically effective amount of cyclophosphamide and a modified T cell containing a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13; and a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

In another aspect, the present disclosure provides a method of treating metastatic castration-resistant prostate cancer in a subject in need thereof, the method comprising administering to the subject a lymphodepleting chemotherapy comprising administering to the subject a therapeutically effective amount of cyclophosphamide and administering to the subject a modified T cell comprising a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the amino acid sequence recited in SEQ ID No. 13; and a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

Drawings

The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments taken together with the accompanying drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1A illustrates the results using purified IVT PSMA RNA CAR and the full length PSMA RNA resolved on an agarose gel.

Figure 1B shows the results of electroporation into ND 444T cells using purified PSMA RNA CAR and examination of CAR expression by flow cytometry. The average fluorescence intensity is marked below the graph.

FIG. 1C illustrates PSMA expression. Purified full-length PSMA RNA was electroporated into Nalm6 or K562 cells (middle and right panels). PSMA expression was examined by flow cytometry.

Figure 1D illustrates the results of using a combined pc3.psma single cell clone. Limiting dilution was performed with pc3.psma cells (left panel), seven individual colonies were isolated and pooled as a new cell line, pc3.psma.7sc (right panel). PSMA expression was examined by flow cytometry.

Figure 2A illustrates the results of using various PSMA RNA CAR cultures with tumor cells and performing CD107a assays. Cells were gated by CD 3.

Fig. 2B illustrates the results of using various PSMA RNA CAR cultures with tumor cells and performing luciferase-based CTL assays. The results are reported as percent killing based on luciferase activity in tumor-only wells in the absence of T cells.

Figure 2C shows the results of using various PSMA RNA CAR cultures with tumor cells and performing ELISA assays. (IL-2, left panel; IFN-. gamma., right panel).

Fig. 3A illustrates the results using PSMA Lenti CAR constructed and transferred into primary human T cells (MOI ═ 3). CAR expression was examined by flow cytometry on day 8.

Figure 3B shows the results of using various PSMA Lenti CARs cultured with or without tumor cells and subjected to CD107a assays. Cells were gated by CD 3. Results from day 12 are shown.

Figure 3C shows the results of using various PSMA Lenti CARs cultured with or without tumor cells and performed a luciferase-based CTL assay. The results are reported as percent killing based on luciferase activity in tumor-only wells in the absence of T cells. Results from day 12 are shown.

FIG. 3D illustrates the results of using various PSMA Lenti CAR in culture with PC3 or PC3.PSMA cells and performing ELISA assays (IL-2, left panel; IFN-. gamma., right panel). Results from day 12 are shown.

Figure 4A illustrates the results of using the switch receptors PD1 × ptm. cd28 or pd1.cd28 to each human PSMA Lenti CAR via F2A and into primary human T cells. PD1 and CAR expression were examined by flow cytometry on day 12.

Figure 4B illustrates the results of using a dominant negative (dn) transforming growth factor beta receptor II (TGFR β II) sequence linked to each human PSMA Lenti CAR via T2A. On day 7, Dn-TGFR β II-PSMA CAR transduced T cells were analyzed by flow cytometry.

Fig. 4C illustrates the results of electroporation into pc3.psma cells using various amounts of purified full-length PDL1 RNA and examination of PDL1 expression by flow cytometry at day 13.

Figure 4D shows the results of culturing pc3.PSMA cells electroporated with pc3.PSMA or PDL1 and performing CD107a assays using various PSMA Lenti CARs. Cells were gated by CD 3. Results from day 14 are shown.

Figure 4E shows the results of culturing pc3.PSMA cells electroporated with pc3.PSMA or PDL1 and performing CD107a assays using various PSMA Lenti CARs. Cells were gated by CD 3. Results from day 14 are shown.

Figure 4F shows the results of culturing pc3.PSMA cells electroporated with pc3.PSMA or PDL1 and performing CD107a assays using various PSMA Lenti CARs. Cells were gated by CD 3. Results from day 14 are shown.

Figure 4G shows the results of culturing pc3.PSMA cells electroporated with pc3.PSMA or PDL1 and performing CD107a assays using various PSMA Lenti CARs. Cells were gated by CD 3. Results from day 14 are shown.

Figure 4H shows the results of using various PSMA Lenti CARs cultured with pc3.PSMA cells and performed a luciferase-based CTL assay. The results are reported as percent killing based on luciferase activity in tumor-only wells in the absence of T cells.

Figure 4I shows the results of culturing with various PSMA Lenti CARs and performing ELISA assays with pc3.PSMA or PDL1 electroporated pc3.PSMA cells. (IL-2, top panel; IFN-. gamma., bottom panel).

Figure 5A shows the results of transduction into primary human T cells using the transduction receptor pd1.cd28 linked to each human PSMA Lenti CAR via F2A. PD1 and CAR expression were examined by flow cytometry.

Figure 5B shows the results of ligation to human 2a10 PSMA Lenti CAR via T2A using a dominant negative (dn) TGFR β II sequence. CAR-transduced T cells were analyzed by flow cytometry.

Figure 5C shows the results of using various PSMA Lenti CARs cultured with pc3.psma.7sc cells and performing the CD107a assay. Cells were gated by CD 3.

Figure 5D shows the results of using various PSMA Lenti CARs cultured with PDL1 electroporated pc3.psma.7sc cells and performing CD107a assays. Cells were gated by CD 3.

Figure 5E shows the results of using various PSMA Lenti CARs cultured with tumor cells and performed a luciferase-based CTL assay. The results are reported as percent killing based on luciferase activity in tumor-only wells in the absence of T cells.

Figure 5F shows the results of using various PSMA Lenti CARs cultured with PC3, pc3.psma.7sc or PDL1 electroporated pc3.psma.pdl1 cells and performing ELISA assays. (IL-2, top panel; IFN-. gamma., bottom panel).

FIG. 5G shows the results of PSMA expression using quantitative PCR. Fold change (Δ Δ CT) was normalized to nalm6.cbg cells. For abbreviations see table 1.

Figure 5H shows the results of using various PSMA Lenti CARs cultured with tumor cells or primary human cells and subjected to CD107a assay. Cells were gated by CD 3.

Fig. 5I shows quantitative data for the experiment shown in fig. 5H. HSAEpC human small airway epithelial cells. HPMEC is human lung microvascular endothelial cells.

Figure 5J shows the results of culturing with primary human cells and performing ELISA assays using various PSMA Lenti CARs. (IL-2, left panel; IFN-. gamma., right panel). HREpC human renal epithelial cells. HSAEpC human small airway epithelial cells. HPMEC is human lung microvascular endothelial cells.

FIG. 5K shows 2X10 using a mouse transduced with a Capillaria (click beetle) and injected (i.v.)⁶Results of individual pc3.psma.7sc cells. After 27 days, 2X10⁶One PSMA CAR-T positively transduced T cell was injected into tumor-bearing mice (i.v.). Bioluminescence imaging (BLI) was performed at multiple time points. The minimum mean emittance (radiance) of the upper graph is 5 x10⁵(ii) a The minimum mean emittance of the lower graph is 3 x10⁵。

FIG. 5L illustrates the quantitative mean emittance of FIG. 5K.

FIG. 6 is a schematic representation of the map of the dn-TGFR β II PSMA CAR construct and pTRPE construct.

Figure 7 shows flow cytometric examination of CAR expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS), or 2F5 PSMA CAR co-transduced along with various switch receptors, as indicated.

Figure 8 shows flow cytometric examination of PD1 and Tim3 expression in T cells transduced with 2F5 PSMA CAR alone (2F5 ICOS), or 2F5 PSMA CAR co-transduced along with various switch receptors, as indicated.

Figure 9 is a graph depicting CD107a expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR along with various switch receptors, as indicated. UTD means not transduced.

Figure 10 is a graph depicting granzyme B expression in T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR along with various switch receptors, as indicated. UTD means not transduced.

Figure 11A is a graph depicting IL-2 secretion by T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR along with various switch receptors, as indicated. NTD means not transduced.

Figure 11B is a graph depicting IFN γ secretion by T cells transduced with 2F5 PSMA ICOS-CAR alone (ICOS), PSMA 41BB-CAR alone (41BB), or 2F5 PSMA CAR along with various switch receptors, as indicated. UTD means not transduced.

Figure 12A is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 12B is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 13 is a graph depicting tumor size in NSG mice tumor-induced by PC-loaded 3-psma. cbg treated with T cells transduced according to the instructions.

Figure 14A is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with indicator transduced T cells.

Figure 14B is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 14C is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 14D is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 14E is a graph depicting quantification of bioluminescence obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Figure 14F is a graph depicting quantification of bioluminescence (left) and tumor size (right) obtained from imaging of PC3-psma. cbg tumor-bearing NSG mice treated with T cells transduced according to the instructions.

Fig. 14G is a table listing T cells transduced in order of tumor control ability from top to bottom according to the instructions. ICOS^YMNMSuperior to WT ICOS. When having ICOSz or ICOSzYMNM, PD1 BB was better than PD1 CD 28.

FIG. 15A is a graph showing that CART-PSMA-TGF β Rdn cells (dnTGFBR2-T2A-Pbbz) demonstrate enhanced antigen-specific proliferation after repeated stimulation (arrows) with PSMA-expressing tumor cells during 42 days of co-culture compared to CART-PSMA (Pbbz). CD19-BBz CART (19BBz) and transduced T cells (mock) were used as controls.

FIG. 15B is a graph showing the mean emittance detected in tumor-bearing mice up to 27 days after T cell injection with CART-PSMA-TGF β Rdn cells (dnTGFBR2-T2A-Pbbz), CART-PSMA cells (Pbbz), and untransduced cells (mock) as a control group.

Fig. 15C is a photograph showing the location and system burden of tumors undergoing weekly bioluminescence imaging (BLI) assessment.

Figure 16 illustrates the study protocol used in the phase 1 clinical trial.

Figure 17 is a graph showing the assessment of CAR-T cell dynamics in subjects via qPCR of CART-PSMA-TGF β Rdn DNA. Subjects 32816-02, -04 and-05 were in group 1, and subjects 32816-06, -07 and-08 were in group 2.

Figure 18A is a graph showing significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFN γ) and ferritin, which were associated with a grade 3 cytokine release syndrome event in subject 32816-06.

Figure 18B is a graph showing significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFN γ) and ferritin, which were associated with a grade 3 cytokine release syndrome event in subject 32816-07.

Figure 19 is a graph showing Prostate Specific Antigen (PSA) responses in group 1 and group 2 patients.

Figure 20A is a graph showing expression of PSMA-TGF β RDN CART (left y-axis, copy number of genomic DNA/ug) and levels of IL-6 (right y-axis, pg/ml) in subject 32816-07, indicating cytokine release syndrome exhibited in the subject one day after infusion.

FIG. 20B is a graph showing cytokine release syndrome management with transient PSA reduction as measured by C-reactive protein (CRP; left y-axis, mg/L) levels and serum ferritin levels (right y-axis, ng/L).

Fig. 21 is a graph showing the number of PSMA-positive Circulating Tumor Cells (CTCs) detected over time in each subject.

Detailed Description

The present invention provides compositions and methods of modified immune cells, such as T cells and NK cells, or their precursor cells, such as modified T cells, that include a Chimeric Antigen Receptor (CAR). In some embodiments, the CAR comprises a Prostate Specific Membrane Antigen (PSMA) binding domain (PSMA-CAR) and has affinity for PSMA on a target cell (e.g., a prostate cancer cell). In some embodiments, the modified immune cell comprises a PSMA-CAR comprising a murine PSMA binding domain. In some embodiments, the modified immune cell comprises a PSMA-CAR comprising a human PSMA-binding domain. Methods of producing such genetically engineered cells are also provided. In some embodiments, the cells and compositions can be used in adoptive cell therapy (adoptive cell therapy), such as adoptive tumor immunotherapy.

In some embodiments, provided immune cells include additional receptors, such as dominant negative receptors and/or switch receptors, to enhance the efficacy of the immune cells in the tumor microenvironment. Such cells are capable of altering or reducing the effects of immunosuppressive signals in the tumor microenvironment. The modified immune cells of the invention counteract the up-regulation and/or expression of inhibitory receptors or ligands that can negatively control T cell activation and T cell function. For example, expression of certain immune checkpoint proteins (e.g., PD-1 or PD-L1) on T cells and/or in the tumor microenvironment can reduce the efficacy and efficacy of adoptive T cell therapy. For example, expression of TGF- β on T cells and/or in the tumor microenvironment may reduce the efficacy and efficacy of adoptive T cell therapy. In the context of adoptive cell therapy, such immunosuppressive signals may otherwise impair certain desired effector functions. Tumor cells and/or cells in the tumor microenvironment typically upregulate immunosuppressive proteins, such as PD-L1, delivering immunosuppressive signals. Such immunosuppressive proteins may also be upregulated on T cells in the tumor microenvironment, e.g., on tumor infiltrating T cells, which may occur upon signaling through antigen receptors or some other activation signal. Such events may contribute to genetically engineered immune cells (e.g., targeting PSMA) T cells acquiring a depleted phenotype, which in turn may lead to reduced function, for example, when present in the vicinity of other cells expressing such proteins. Thus, the modified immune cells of the invention address T cell depletion and/or lack of T cell persistence (persistence), which is an obstacle to the efficacy and therapeutic outcome of conventional adoptive cell therapies.

The invention includes PSMA CARs and their use in treating cancer. In certain embodiments, the invention encompasses human PSMACAR having a dominant negative receptor and/or a switch receptor. One of the major obstacles to cancer immunotherapy is the tumor microenvironment. Upregulation of immunosuppressive molecules (e.g., PD-1) negatively regulates T cell activity.

The present invention is based on the following findings: t cells comprising a PSMA-CAR and a dominant negative receptor and/or switch receptor are able to bypass the role of immunosuppressive molecules in the tumor microenvironment, providing continuous and effective anti-tumor activity.

It is to be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Furthermore, unless otherwise indicated, the experiments described herein employ conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are known to those skilled in the art and are explained fully in the literature. See, e.g., Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987. Across. 2008), including all supplements, Molecular Cloning: A Laboratory Manual (fourth edition) by MR Green and J.Sambrook and Harlow et al, Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013,2 edition).

A. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The definitions provided herein override any dictionary or foreign definitions if any potential ambiguity exists. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The terms "including" and other forms of words such as "includes" and "included" are non-limiting.

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature and laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

The following definitions of selected terms may facilitate understanding of the present disclosure.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, "about," when referring to a measurable value such as an amount, time period, or the like, is meant to encompass a variation of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from a given value, provided such variation is suitable for practicing the disclosed methods.

"activation," as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" or the like refers to a T cell that undergoes cell division.

As used herein, the term "alleviating" a disease refers to reducing the severity of one or more symptoms of the disease.

The term "antibody", as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural source or from a recombinant source, and can be an immunoreactive portion of an intact immunoglobulin (e.g., a binding fragment of an antibody). Antibodies are typically tetramers of immunoglobulin molecules. The Antibodies of the invention may exist In a variety of forms including, for example, polyclonal, monoclonal, Fv, Fab and F (ab)2, as well as single chain Antibodies (scFv) and humanized Antibodies (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl.Acad.Sci.USA 85: 5879-.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.

"antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

"antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation, and alpha and beta light chains refer to the two major antibody light chain isotypes.

The term "synthetic antibody" as used herein, refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to refer to antibodies that have been produced by synthesis of a DNA molecule encoding the antibody and which expresses the antibody protein or specifies the amino acid sequence of the antibody, which DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and widely known in the art.

The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. The immune response may involve antibody production, or activation of specific immune competent cells, or both. The skilled person will appreciate that any macromolecule, including virtually all proteins or peptides, may be used as an antigen.

Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled person will understand that any DNA, which comprises a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, thus encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit the desired immune response. Moreover, the skilled artisan will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that the antigen may be produced, synthesized or may be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

As used herein, the term "autologous" refers to any substance derived from the same individual that is subsequently reintroduced into the individual. "allogenic" refers to any substance derived from different animals of the same species. "xenogeneic" refers to any substance derived from animals of different species.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor engineered to be expressed on immune cells and specifically bind to an antigen. The CAR can be used as an adoptive cell transfer therapy. The T cells are removed from the patient and modified to express a receptor specific for the antigen or a particular form of the antigen. In some embodiments, the CAR is specific for a selected target, such as a cell expressing a prostate-specific membrane antigen. The CAR can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain, the extracellular domain including a tumor-associated antigen binding region.

The term "co-stimulatory ligand" as used herein includes molecules on antigen presenting cells (e.g., artificial apc (aapc), dendritic cells, B cells, etc.) that specifically bind to cognate co-stimulatory molecules on T cells, thereby providing signals mediating T cell responses including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, binding of the TCR/CD3 complex to peptide-loaded MHC molecules. Costimulatory ligands can include, but are not limited to, CD7, B7-1(CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include, in particular, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphofunctionally related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD 83.

"costimulatory molecule" refers to an associated binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

As used herein, "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR/CD3 ligation), results in the up-or down-regulation of T cell proliferation and/or key molecules.

A "disease" is a state of health of an animal in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate if the disease is not ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is less favorable than if it were not in the disorder. Remaining untreated, the condition does not necessarily cause a further reduction in the health status of the animal.

As used herein, the term "downregulating" refers to reducing or eliminating gene expression of one or more genes.

The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to a compound, formulation, material or composition as described herein that is effective to achieve a particular biological result or to provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, amounts of the composition that cause a detectable level of immunosuppression or immune tolerance when administered to a mammal as compared to an immune response detected in the absence of the composition of the present invention. Immune responses can be readily assessed in a number of art-recognized ways. One skilled in the art will appreciate that the amount of the composition administered herein varies and can be readily determined based on a number of factors, such as the disease or disorder being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, and the particular compound being administered, among others.

The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (e.g., rRNA, tRNA, and mRNA) or defined amino acid sequences, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for transcription of a gene or cDNA) may be referred to as encoding a protein or other product of the gene or cDNA.

As used herein, the term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

As used herein, the term "epitope" is defined as a small chemical molecule on an antigen that elicits an immune response, inducing a B and/or T cell response. An antigen may have one or more epitopes. Most antigens have multiple epitopes; that is, these antigens are multivalent. Typically, epitopes are about 10 amino acids and/or sugars in size. Preferably, the epitope is about 4 to 18 amino acids, more preferably about 5 to 16 amino acids, and even more preferably 6 to 14 amino acids, more preferably about 7 to 12 amino acids, and most preferably about 8 to 10 amino acids. It will be appreciated by those skilled in the art that, in general, the primary condition for antigen specificity is the overall three-dimensional structure of the molecule, rather than its specific linear sequence, and thus the differentiation of different epitopes. Based on the present disclosure, the peptides of the invention may be epitopes.

As used herein, the term "exogenous" refers to any substance introduced from or produced outside of an organism, cell, tissue, or system.

As used herein, the term "expansion" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the number of ex vivo expanded T cells is increased relative to the number of culture media in which it was originally present. In another embodiment, the number of ex vivo expanded T cells is increased relative to other cell types in the culture medium. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and propagated outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

An "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or included in liposomes) and viruses (e.g., sendai, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab ', F (ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (e.g., mouse, rat or rabbit) (donor antibody) having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further improve and optimize antibody performance. Typically, a humanized antibody will comprise substantially all of the following: at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details see Jones et al, Nature,321:522-525, 1986; reichmann et al, Nature,332: 323-; presta, curr, Op, struct, biol.,2: 593-. "fully human" refers to an immunoglobulin, such as an antibody or binding fragment thereof, in which the entire molecule is of human origin or consists of an amino acid sequence that is identical to the human form of the antibody.

As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Five members included in this class of proteins are IgA, IgG, IgM, IgD and IgE. IgA is a primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced in the primary immune response of most subjects. It is the most effective immunoglobulin in agglutination, complement fixation and other antibody responses, and is important in combating bacteria and viruses. IgD is an immunoglobulin that does not have known antibody function but can be used as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to allergen.

As used herein, the term "identity" refers to a subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules, for example between two polypeptide molecules. When two amino acid sequences have identical residues at the same position, e.g., if two polypeptide molecules in each position is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have identical residues in the same aligned position is typically expressed in terms of percentage. Identity between two amino acid sequences is a direct function of the number of matched or identical positions, e.g., if half of the positions in two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce antibody formation and/or activate lymphocytes to remove the antigen.

As used herein, the term "immunosuppression" refers to a reduction in the overall immune response.

The term "isolated" means altered or removed from the native state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely separated from the coexisting materials of its natural state, is "isolated. An isolated nucleic acid or protein can be present in a substantially purified form, or can be present in a non-natural environment (such as, for example, a host cell).

As used herein, "lentivirus" refers to a genus of the family retroviridae. Among retroviruses, lentiviruses are the only capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of the host cell, so they are one of the most efficient methods of gene delivery vectors. HIV, S1V and FIV are examples of lentiviruses. Lentivirus-derived vectors provide a means to accomplish significant levels of gene transfer in vivo.

As used herein, the term "modified" refers to a change in the state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by introducing nucleic acids.

As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject as compared to the level of a response in a subject in the absence of the treatment or compound, and/or as compared to the level of a response in an otherwise identical but untreated subject. The term includes disrupting and/or affecting the natural signal or response, thereby mediating a beneficial therapeutic response in a subject (preferably a human).

In the context of the present invention, the following abbreviations are used for the commonly occurring nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may include an intron(s) in some versions.

The term "operably linked" or "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence.

The term "polynucleotide" as used herein is defined as a strand of nucleotides. In addition, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. Nucleic acids are generally known to those skilled in the art as polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques, PCR, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains, such as are also commonly referred to in the art as peptides, oligopeptides and oligomers, and also to longer chains, which are commonly referred to in the art as proteins, of which there are many types. "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, this cross-species reactivity does not itself alter the specific class of antibodies. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity does not itself alter the specific class of antibodies. In some instances, the term "specific binding" or "specifically binds" may be used with reference to the interaction of an antibody, protein or peptide with a second chemical species, to indicate that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to specific protein structures rather than proteins in general. If the antibody is specific for epitope "A", the presence of a molecule comprising epitope A (or free, unlabeled A) in a reaction comprising labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "stimulation" refers to a primary response induced by binding of a stimulating molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as down-regulation of TGF- β and/or reorganization of cytoskeletal structures, and the like.

"stimulatory molecule," as the term is used herein, refers to a molecule on a T cell that specifically binds to an cognate stimulatory ligand present on an antigen presenting cell.

As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., aAPC, dendritic cell, B-cell, etc.), can specifically bind to an associated binding partner on a T cell (referred to herein as a "stimulatory molecule"), thereby mediating a primary response of the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, inter alia, MHC class I molecules loaded with peptides, anti-CD 3 antibodies, superagonist anti-CD 28 antibodies, and superagonist anti-CD 2 antibodies.

The term "subject" is intended to include living organisms (e.g., mammals) within which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, domestic animals and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

"target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid that will specifically bind to a binding molecule under conditions sufficient for binding to occur.

The term "therapeutic" as used herein means treatment and/or prevention. The therapeutic effect is obtained by inhibition, alleviation or eradication of the disease state.

"transplant" refers to a biocompatible lattice (biocompatible lattice) or donor tissue, organ or cell to be transplanted. Examples of grafts may include, but are not limited to, skin cells or tissues, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Graft may also mean any material to be administered to a host. For example, a graft may mean a nucleic acid or protein.

As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. Cells include cells of a primary subject and progeny thereof.

As used herein, the term "treating" refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

A "vector" is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, sendai viral vectors, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

The range is as follows: throughout this disclosure, various aspects of the invention may be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range from 1 to 6 should be considered to have certain disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the size range.

B. Chimeric antigen receptors

The present invention provides compositions and methods of modified immune cells or precursors thereof (e.g., modified T cells), including Chimeric Antigen Receptors (CARs). Thus, in some embodiments, the immune cell has been genetically modified to express the CAR. The CAR of the invention comprises an antigen binding domain, a transmembrane domain, a hinge domain, and an intracellular signaling domain.

The antigen binding domain may be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain described elsewhere herein, for expression in a cell. In one embodiment, the first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third nucleic acid sequence encoding an intracellular domain.

The antigen binding domains described herein may be combined with any transmembrane domain described herein, any intracellular or cytoplasmic domain described herein, or any other domain described herein that may be included in a CAR of the invention. The subject CARs of the invention may also include a spacer domain as described herein. In some embodiments, the antigen binding domain, transmembrane domain, and intracellular domain are separated by a linker.

Antigen binding domains

The antigen binding domain of the CAR is the extracellular region of the CAR for binding a specific target antigen, including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises an affinity for a target antigen on a target cell. The target antigen may include any type of protein or epitope thereof associated with the target cell. For example, the CAR can include an affinity for a target antigen on a target cell that is indicative of a particular disease state of the target cell.

In an exemplary embodiment, the target cell antigen is Prostate Specific Membrane Antigen (PSMA). PSMA is a membrane-bound protein expressed on the cell surface and is reported to be highly overexpressed in prostate cancer tissues. PSMA expression is directly related to the progression of tumor grade and stage and is thought to confer a selective growth advantage on prostate cancer cells. As such, exemplary CARs of the present disclosure are specific for PSMA on target cells.

As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell can include a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target-specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In exemplary embodiments, the CARs of the present disclosure having affinity for PSMA on a target cell can comprise a PSMA binding domain. In some embodiments, the PSMA-binding domain is a murine PSMA-binding domain, e.g., the PSMA-binding domain is of murine origin. In some embodiments, the PSMA-binding domain is a human PSMA-binding domain, e.g., the PSMA-binding domain is of human origin.

In some embodiments, a CAR of the present disclosure can have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR can have affinity for one or more target antigens on a target cell. In such embodiments, the CAR is a bispecific CAR, or a multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR that includes one or more target-specific binding domains with affinity for the same target antigen can bind to different epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, the binding domains may be arranged in tandem and may be separated by a linker peptide. For example, in a CAR comprising two target-specific binding domains, the binding domains are covalently linked to each other on a single polypeptide chain by an oligopeptide or polypeptide linker, an Fc hinge region, or a membrane hinge region.

In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single chain variable fragment (scFv). In some embodiments, the PSMA-binding domain of the present invention is selected from the group consisting of a PSMA-specific antibody, a PSMA-specific Fab, and a PSMA-specific scFv. In one embodiment, the PSMA-binding domain is a PSMA-specific antibody. In one embodiment, the PSMA-binding domain is a PSMA-specific Fab. In one embodiment, the PSMA-binding domain is a PSMA-specific scFv.

The antigen binding domain may include any domain that binds to an antigen and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antigens, human antibodies, humanized antibodies, non-human antibodies, and any fragments thereof. In some embodiments, the antigen binding domain portion comprises a mammalian antibody or fragment thereof. The choice of antigen binding domain may depend on the type and number of antigens present on the surface of the target cell.

As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:: VL heterodimer. The heavy (VH) and light (VL) chains are joined either directly or via a peptide-encoding linker which links the N-terminus of the VH to the C-terminus of the VL, or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., PSMA-binding domain) comprises an scFv having the configuration of VH-linker-VL from N-terminus to C-terminus. In some embodiments, the antigen-binding domain (e.g., PSMA-binding domain) comprises an scFv having the configuration VL-linker-VH from N-terminus to C-terminus. Those skilled in the art will be able to select an appropriate configuration for use in the present invention.

The linker is generally glycine rich for flexibility and serine or threonine rich for solubility. The linker being capable of linking to the extracellular antigen-binding domainA heavy chain variable region and a light chain variable region. Non-limiting examples of linkers are disclosed in Shen et al, anal. chem.80(6):1910- > 1917(2008) and WO 2014/087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, Glycine Serine (GS) linkers, such as (GS)_n、(GSGGS)_n(SEQ ID NO:1)、(GGGS)_n(SEQ ID NO:2) and (GGGGS)_n(SEQ ID NO:3) wherein n represents an integer of at least 1. Exemplary linker sequences may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:4), GGSGG (SEQ ID NO:5), GSGSGSG (SEQ ID NO:6), GSGGG (SEQ ID NO:7), GGGSG (SEQ ID NO:8), GSSSG (SEQ ID NO:9), GGGGS (SEQ ID NO:10), GGGGSGGGGSGGGGS (SEQ ID NO:11), and the like. One skilled in the art will be able to select an appropriate linker sequence for use in the present invention. In one embodiment, an antigen binding domain of the invention (e.g., a PSMA binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and VL are separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGS (SEQ ID NO:11), which may be encoded by nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO: 12).

Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies may be expressed from nucleic acids comprising VH-and VL-encoding sequences as described in Huston, et al (Proc. Nat. Acad. Sci. USA,85: 5879-. See also U.S. patent nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. patent publication nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al, Hyrbidoma (Larchmt) 200827 (6): 455-51; Peter et al, J Cachexia Musclle 2012 augst 12; Shieh et al, J Imunol 2009183 (4): 2277-85; Giomarelli et al, Thromb Haemost 200797 (6): 955-63; Fife et al, J Clin Invst 2006116 (8): 2252-61; Brocks et al, Immunotechnology 19973 (3): 173-84; Moosmayer et al, Thermus 19952 (10: 31-40)). Competitive (Agonitic) scFv with stimulatory activity have been described (see, e.g., Peter et al, J Bio Chem 200325278(38): 36740-7; Xie et al, Nat Biotech 199715 (8): 768-71; Ledbetter et al, Crit Rev Immunol 199717 (5-6): 427-55; Ho et al, BioChim Biophys Acta 20031638 (3): 257-66).

As used herein, "Fab" refers to a fragment of an antibody structure that binds an antigen but is monovalent and does not have an Fc portion, e.g., a papain digested antibody produces two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind to an antigen).

As used herein, "F (ab ') 2" refers to an antibody fragment produced by pepsin digestion of an intact IgG antibody, wherein the fragment has two antigen-binding (ab ') (bivalent) regions, wherein each (ab ') region comprises two independent amino acid chains, a portion of the H chain and a light chain (L) are connected by an S-S bond to bind to an antigen, and wherein the remaining H chains are then connected together. The "F (ab ') 2" fragment can be divided into two separate Fab' fragments.

In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR can include a human antibody, or a fragment thereof, as described elsewhere herein.

In exemplary embodiments, the PSMA-CARs of the invention comprise a PSMA binding domain, e.g., a PSMA-specific scFv.

(a) Murine PSMA binding domains and variants thereof.

In certain embodiments, the PSMA-CARs of the invention comprise a murine PSMA binding domain or variant thereof.

In certain embodiments, a PSMA-CAR of the invention comprises a PSMA binding domain of a non-human PSMA antibody (e.g., a mouse or rat PSMA antibody), or a variant thereof. It is well known in the art that murine or other non-human antibodies can be raised by immunizing a non-human (e.g., mouse) with human PSMA or a fragment thereof.

In one embodiment, the PSMA binding domain is a murine J591PSMA binding domain, which is included in the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:14)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:15)。

one skilled in the art knows the tolerance of the murine J591PSMA binding domain (Tolerable variants) while maintaining binding to PSMA. For example, in some embodiments, a PSMA-binding domain is a murine J591 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a murine J591 PSMA-binding domain amino acid sequence included in SEQ ID No. 14. In one embodiment, the PSMA binding domain is the murine J591PSMA binding domain included in the amino acid sequence set forth in SEQ ID NO. 14.

In some embodiments, the PSMA binding domain is a murine J591PSMA binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a murine J591PSMA binding domain encoding sequence included in SEQ ID No. 15. In one embodiment, the PSMA binding domain is the murine J591PSMA binding domain encoded by the coding sequence included in the nucleic acid sequence set forth in SEQ ID NO. 15.

In exemplary embodiments, the PSMA-CARs of the invention comprise a PSMA binding domain, e.g., a PSMA-specific scFv. In one embodiment, the PSMA binding domain is a murine J591PSMA binding domain comprising the amino acid sequence set forth below:

DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:13)，

it may be encoded by the nucleic acid sequences set forth below:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:180)。

one skilled in the art knows the allowable variations of the murine J591PSMA binding domain while maintaining binding to human PSMA. For example, in some embodiments, the PSMA binding domain is a murine J591PSMA binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 13. In one embodiment, the PSMA binding domain is a murine J591PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO 13.

In some embodiments, the PSMA binding domain is a murine J591PSMA binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 180. In one embodiment, the PSMA binding domain is the murine J591PSMA binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 180.

In one embodiment, the murine J591PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVP DRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLK(SEQ ID NO:16)，

it may be encoded by the nucleic acid sequences set forth below:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAA(SEQ ID NO:17)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human RPSMA. For example, in some embodiments, a murine J591PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 16. In one embodiment, the murine J591PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16.

In some embodiments, a murine J591 PSMA binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 17. In one embodiment, the murine J591 PSMA binding domain comprises the light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 17.

In one embodiment, the murine J591 PSMA binding domain comprises the light chain variable region described in NCBI GenBank sequence database ID: CCA78125.1, comprising the amino acid sequence set forth below:

DIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLAITNVQSEDLADYFCQQYNSYPLTFGAGTKLEIKR(SEQ ID NO:181)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a murine J591 PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 181. In one embodiment, the murine J591 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 181. The light chain variable region of the murine J591 PSMA binding domain includes three light chain Complementarity Determining Regions (CDRs). As used herein, "complementarity determining region" or "CDR" refers to the region of the variable chain of an antigen binding molecule that binds to a specific antigen. Thus, the murine J591 PSMA binding domain can comprise a light chain variable region comprising the CDR1 represented by amino acid sequence KASQDVGTAVD (SEQ ID NO: 18); CDR2 represented by the amino acid sequence WASTRHT (SEQ ID NO: 19); and CDR3 represented by amino acid sequence QQYNSYPLT (SEQ ID NO: 20). Those skilled in the art know the allowable variation of the CDRs of the light chain while maintaining their contribution to binding of PSMA. For example, a murine J591 PSMA binding domain may comprise a light chain variable region comprising CDR1, the CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO. 18. For example, a murine J591 PSMA binding domain may comprise a light chain variable region comprising CDR2, the CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO 19. For example, a murine J591 PSMA binding domain may comprise a light chain variable region comprising CDR3, the CDR3 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the murine J591 PSMA binding domain comprises a light chain variable region comprising three of the foregoing light chain variable region CDRs.

In one embodiment, the murine J591 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:21)，

it may be encoded by the nucleic acid sequences set forth below:

GAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC(SEQ ID NO:22)。

those skilled in the art are aware of the allowable variations of the heavy chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a murine J591 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 21. In one embodiment, the murine J591 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21.

In some embodiments, a murine J591 PSMA binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 22. In one embodiment, the murine J591 PSMA binding domain comprises the heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 22.

In one embodiment, the murine J591 PSMA binding domain comprises the heavy chain variable region described in NCBI GenBank sequence database ID: CCA78124.1, comprising the amino acid sequence set forth below:

EVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSS(SEQ ID NO:182)。

those skilled in the art know the allowable variations of the heavy chain variable region while maintaining its contribution to binding of PSMA. For example, in some embodiments, a murine J591 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 182. In one embodiment, the murine J591 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 182.

The heavy chain variable region of murine J591 PSMA binding domain includes three heavy chain Complementarity Determining Regions (CDRs). Thus, the murine J591 PSMA binding domain can comprise a heavy chain variable region comprising the CDR1 represented by amino acid sequence GYTFTEYTIH (SEQ ID NO: 23); CDR2 represented by amino acid sequence NINPNNGGTTYNQKFED (SEQ ID NO: 24); and a CDR3 represented by the amino acid sequence GWNFDY (SEQ ID NO: 25). Those skilled in the art are aware of the allowable variations of the CDRs of the heavy chain while maintaining their contribution to binding of human PSMA. For example, a murine J591 PSMA binding domain may comprise a heavy chain variable region comprising CDR1, the CDR1 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID NO: 23. For example, a murine J591 PSMA binding domain may comprise a heavy chain variable region comprising CDR2, the CDR2 comprising an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO. 24. For example, a murine J591 PSMA binding domain may comprise a heavy chain variable region comprising CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID NO: 25. In one embodiment, the murine J591 PSMA binding domain comprises a heavy chain variable region comprising three of the foregoing CDRs of the heavy chain variable region.

In one embodiment, the PSMA binding domain is a murine J591 PSMA binding domain comprising an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs 16 and 21.

In one embodiment, the PSMA binding domain is a murine J591 PSMA binding domain comprising an amino acid sequence comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NOs 181 and 182.

(b) Humanized PSMA binding domains

In certain embodiments, the PSMA-CARs of the invention comprise a humanized variant of the PSMA binding domain of a non-human PSMA antibody or a variant or fragment thereof. In certain exemplary embodiments, the PSMA CAR comprises a humanized variant of a murine J591 antibody that binds human PSMA. Methods of humanizing murine antibodies are known in the art.

In one embodiment, the PSMA-binding domain is a humanized PSMA-specific binding domain. In certain embodiments, the PSMA binding domain is a humanized J591 PSMA binding domain. In certain embodiments, the PSMA-binding domain comprises any of the heavy and light chain variable regions disclosed in PCT publication nos. WO2017212250a1 and WO2018033749a1, the disclosures of which are incorporated herein by reference in their entirety. For example, the PSMA-binding domain of the present invention may comprise an scFv comprising any of the heavy and light chain variable regions disclosed therein. Thus, the PSMA-CARs of the present invention include humanized variants of the murine J591 antibody that bind human PSMA disclosed in PCT publication nos. WO2017212250a1 and WO2018033749a 1.

In certain embodiments, the PSMA-binding domain of the present invention may comprise the heavy chain variable region and the light chain variable region of any of those recited in table 19:

TABLE 19 humanized PSMA binding heavy and light chain variable sequences

(c) Human PSMA binding domains

In certain embodiments, the PSMA-CARs of the invention comprise the PSMA binding domain of a human PSMA antibody or variant thereof. In one embodiment, the PSMA-binding domain is a human 1C3 PSMA-binding domain comprising the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK(SEQ ID NO:26)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:27)。

those skilled in the art are aware of the allowable variations of human 1C3PSMA while maintaining binding to human PSMA. For example, in some embodiments, the PSMA-binding domain is a human 1C3 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 26. In one embodiment, the PSMA binding domain is a human 1C3PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO. 26.

In some embodiments, the PSMA-binding domain is a human 1C3 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 27. In one embodiment, the PSMA binding domain is the human 1C3 PSMA binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO. 27.

In one embodiment, the human 1C3 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

PQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSS(SEQ ID NO:28)，

it may be encoded by the nucleic acid sequences set forth below:

CCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO:29)。

those skilled in the art are aware of the allowable variations of the heavy chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 1C3 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 28. In one embodiment, the human 1C3 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 28.

In some embodiments, the human 1C3PSMA binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 29. In one embodiment, the human 1C3PSMA binding domain comprises the heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 29.

The heavy chain variable region of the human 1C3PSMA binding domain includes three heavy chain Complementarity Determining Regions (CDRs). Thus, the human 1C3PSMA binding domain may comprise a heavy chain variable region comprising CDR1 represented by the amino acid sequence SYAMH (SEQ ID NO: 30); CDR2 represented by amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO: 31); and CDR3 represented by amino acid sequence AVPWGSRYYYYGMDV (SEQ ID NO: 32). Those skilled in the art know the allowable variations of the CDRs of the heavy chain while maintaining their contribution to binding of PSMA. For example, a human 1C3PSMA binding domain can comprise a heavy chain variable region comprising a CDR1, which CDR1 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 30. For example, a human 1C3PSMA binding domain can comprise a heavy chain variable region comprising a CDR2, which CDR2 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 31. For example, a human 1C3PSMA binding domain can comprise a heavy chain variable region comprising a CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 32. In one embodiment, the human 1C3PSMA binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

In one embodiment, the human 1C3 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK (SEQ ID NO:33), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:34)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 1C3 PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 33. In one embodiment, the human 1C3 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33.

In some embodiments, the human 1C3PSMA binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 34. In one embodiment, the human 1C3PSMA binding domain comprises the light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 34.

The light chain variable region of the human 1C3PSMA binding domain includes three light chain Complementarity Determining Regions (CDRs). Thus, the human 1C3PSMA binding domain can comprise a light chain variable region comprising CDR1 represented by amino acid sequence RASQGISSALA (SEQ ID NO: 35); CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 36); and CDR3 represented by amino acid sequence QQFNSYPLT (SEQ ID NO: 37). Those skilled in the art know the allowable variation of the CDRs of the light chain while maintaining their contribution to binding of PSMA. For example, a human 1C3PSMA binding domain can comprise a light chain variable region comprising a CDR1, which CDR1 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 35. For example, a human 1C3PSMA binding domain can comprise a light chain variable region comprising a CDR2, which CDR2 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 36. For example, a human 1C3PSMA binding domain can comprise a light chain variable region comprising a CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 37. In one embodiment, the human 1C3PSMA binding domain comprises a light chain variable region comprising three of the foregoing light chain variable region CDRs.

In one embodiment, the PSMA binding domain is a human 2a10PSMA binding domain comprising the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK(SEQ ID NO:38)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:39)。

those skilled in the art are aware of the allowable variations of human 2a10PSMA while maintaining binding to human PSMA. For example, in some embodiments, the PSMA binding domain is a human 2a10PSMA binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 38. In one embodiment, the PSMA binding domain is a human 2A10PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 38.

In some embodiments, the PSMA-binding domain is a human 2a10 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 39. In one embodiment, the PSMA binding domain is a human 2A10PSMA binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 39.

In one embodiment, the human 2a10 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSS(SEQ ID NO:40)，

it may be encoded by the nucleic acid sequences set forth below:

CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA(SEQ ID NO:41)。

those skilled in the art are aware of the allowable variations of the heavy chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2a10 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 40. In one embodiment, the human 2A10 PSMA binding domain includes a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 40.

In some embodiments, the human 2a10 PSMA binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 41. In one embodiment, the human 2A10 PSMA binding domain comprises the heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 41.

The heavy chain variable region of the human 2a10 PSMA binding domain includes three heavy chain Complementarity Determining Regions (CDRs). Thus, the human 2A10 PSMA binding domain can include a heavy chain variable region comprising CDR1 represented by the amino acid sequence SNWIG (SEQ ID NO: 42); CDR2 represented by amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 43); and CDR3 represented by amino acid sequence QTGFLWSSDL (SEQ ID NO: 44). Those skilled in the art are aware of the allowable variations of the CDRs of the heavy chain while maintaining their contribution to binding of human PSMA. For example, a human 2a10 PSMA binding domain can include a heavy chain variable region comprising a CDR1, which CDR1 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 42. For example, a human 2a10 PSMA binding domain can include a heavy chain variable region comprising a CDR2, which CDR2 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 43. For example, a human 2a10 PSMA binding domain can include a heavy chain variable region comprising a CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 44. In one embodiment, the human 2a10 PSMA binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

In one embodiment, the human 2a10 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK (SEQ ID NO:45), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA(SEQ ID NO:46)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2a10 PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 45. In one embodiment, the human 2A10 PSMA binding domain comprises a light chain variable region binding domain comprising the amino acid sequence set forth in SEQ ID NO: 45.

In some embodiments, the human 2a10 PSMA binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 46. In one embodiment, the human 2A10 PSMA binding domain comprises the light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 46.

The light chain variable region of the human 2a10 PSMA binding domain includes three light chain Complementarity Determining Regions (CDRs). Thus, the human 2A10 PSMA binding domain can include a light chain variable region comprising the CDR1 represented by amino acid sequence CRASQDISSAL (SEQ ID NO: 47); CDR2 represented by the amino acid sequence YDASSLES (SEQ ID NO: 48); and CDR3 represented by amino acid sequence CQQFNSYPLT (SEQ ID NO: 49). Those skilled in the art know the allowable variation of the CDRs of the light chain while maintaining their contribution to binding of PSMA. For example, a human 2a10 PSMA binding domain can include a light chain variable region comprising a CDR1, which CDR1 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 47. For example, a human 2a10 PSMA binding domain can include a light chain variable region comprising a CDR2, which CDR2 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 48. For example, a human 2a10 PSMA binding domain can include a light chain variable region comprising a CDR3, which CDR3 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 49. In one embodiment, the human 2a10 PSMA binding domain comprises a light chain variable region comprising three of the foregoing light chain variable region CDRs.

In one embodiment, the PSMA-binding domain is a human 2F5 PSMA-binding domain comprising the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIK(SEQ ID NO:50)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAA(SEQ ID NO:51)。

those skilled in the art are aware of the allowable variations of human 2F5PSMA while maintaining binding to human PSMA. For example, in some embodiments, the PSMA binding domain is a human 2F5PSMA binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 50. In one embodiment, the PSMA binding domain is a human 2F5PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 50.

In some embodiments, the PSMA-binding domain is a human 2F5 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 51. In one embodiment, the PSMA binding domain is the human 2F5PSMA binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 51.

In one embodiment, the human 2F5 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

PEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSS(SEQ ID NO:52)，

it may be encoded by the nucleic acid sequences set forth below:

CCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA(SEQ ID NO:53)。

those skilled in the art are aware of the allowable variations of the heavy chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2F5 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 52. In one embodiment, the human 2F5 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 52.

In some embodiments, the human 2F5 PSMA binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 53. In one embodiment, the human 2F5 PSMA binding domain comprises the heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 53.

The heavy chain variable region of the human 2F5PSMA binding domain includes three heavy chain Complementarity Determining Regions (CDRs). Thus, the human 2F5PSMA binding domain may comprise a heavy chain variable region comprising CDR1 represented by SNWIG (SEQ ID NO: 54); CDR2 represented by amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 55); and CDR3 represented by amino acid sequence QTGFLWSFDL (SEQ ID NO: 56). Those skilled in the art know the allowable variations of the CDRs of the heavy chain while maintaining their contribution to binding of PSMA. For example, a human 2F5PSMA binding domain can include a heavy chain variable region comprising a CDR1, which CDR1 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 54. For example, a human 2F5PSMA binding domain can include a heavy chain variable region comprising a CDR2, which CDR2 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 55. For example, a human 2F5PSMA binding domain can include a heavy chain variable region comprising a CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 56. In one embodiment, the human 2F5PSMA binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

In one embodiment, the human 2F5 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

AIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIK (SEQ ID NO:57), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAA(SEQ ID NO:58)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2F5 PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 57. In one embodiment, the human 2F5 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 57.

In some embodiments, the human 2F5PSMA binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 58. In one embodiment, the human 2F5PSMA binding domain comprises the light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 58.

The light chain variable region of the human 2F5PSMA binding domain includes three light chain Complementarity Determining Regions (CDRs). Thus, the human 2F5PSMA binding domain can comprise a light chain variable region comprising CDR1 represented by amino acid sequence RASQDISSALA (SEQ ID NO: 59); CDR2 represented by the amino acid sequence DASSLES (SEQ ID NO: 60); and CDR3 represented by amino acid sequence QQFNSYPLT (SEQ ID NO: 61). Those skilled in the art know the allowable variation of the CDRs of the light chain while maintaining their contribution to binding of PSMA. For example, a human 2F5PSMA binding domain can include a light chain variable region comprising a CDR1, which CDR1 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 59. For example, a human 2F5PSMA binding domain can include a light chain variable region comprising a CDR2, which CDR2 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID NO: 60. For example, a human 2F5PSMA binding domain can include a light chain variable region comprising a CDR3, which CDR3 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 61. In one embodiment, the human 2F5PSMA binding domain comprises a light chain variable region comprising three of the foregoing light chain variable region CDRs.

In one embodiment, the PSMA binding domain is a human 2C6 PSMA binding domain comprising the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFTFGPGTKVDIK(SEQ ID NO:62)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO:63)。

those skilled in the art are aware of the allowable variations of human 2C6 PSMA while maintaining binding to human PSMA. For example, in some embodiments, the PSMA-binding domain is a human 2C6 PSMA-binding domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 62. In one embodiment, the PSMA binding domain is a human 2C6 PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 62.

In some embodiments, the PSMA-binding domain is a human 2C6 PSMA-binding domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 63. In one embodiment, the PSMA binding domain is the human 2C6 PSMA binding domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 63.

In one embodiment, the human 2C6 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth below:

PEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSS(SEQ ID NO:64)，

it may be encoded by the nucleic acid sequences set forth below:

CCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA(SEQ ID NO:65)。

those skilled in the art are aware of the allowable variations of the heavy chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2C6 PSMA binding domain comprises a heavy chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 64. In one embodiment, the human 2C6 PSMA binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 64.

In some embodiments, the human 2C6 PSMA binding domain comprises a heavy chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 65. In one embodiment, the human 2C6 PSMA binding domain comprises the heavy chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 65.

The heavy chain variable region of the human 2C6PSMA binding domain includes three heavy chain Complementarity Determining Regions (CDRs). Thus, the human 2C6PSMA binding domain may comprise a heavy chain variable region comprising CDR1 represented by the amino acid sequence TNYWI (SEQ ID NO: 66); CDR2 represented by amino acid sequence GIIYPGDSDTRYSPSFQG (SEQ ID NO: 67); and CDR3 represented by amino acid sequence SPGYTSSWTS (SEQ ID NO: 68). Those skilled in the art know the allowable variations of the CDRs of the heavy chain while maintaining their contribution to binding of PSMA. For example, a human 2C6PSMA binding domain can include a heavy chain variable region comprising a CDR1, which CDR1 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 66. For example, a human 2C6PSMA binding domain can include a heavy chain variable region comprising a CDR2, which CDR2 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 67. For example, a human 2C6PSMA binding domain can include a heavy chain variable region comprising a CDR3, which CDR3 comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 68. In one embodiment, the human 2C6PSMA binding domain comprises a heavy chain variable region comprising three of the foregoing heavy chain variable region CDRs.

In one embodiment, the human 2C6 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth below:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFTFGPGTKVDIK (SEQ ID NO:69), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO:70)。

those skilled in the art are aware of the allowable variations of the light chain variable region while maintaining its contribution to binding of human PSMA. For example, in some embodiments, a human 2C6 PSMA binding domain comprises a light chain variable region comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 69. In one embodiment, the human 2C6 PSMA binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 69.

In some embodiments, the human 2C6PSMA binding domain comprises a light chain variable region encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 70. In one embodiment, the human 2C6PSMA binding domain comprises the light chain variable region encoded by the nucleic acid sequence set forth in SEQ ID NO: 70.

The light chain variable region of the human 2C6PSMA binding domain includes three light chain Complementarity Determining Regions (CDRs). Thus, the human 2C6PSMA binding domain can comprise a light chain variable region comprising CDR1 represented by amino acid sequence CRASQSVSSYL (SEQ ID NO: 71); consisting of a CDR2 representing the amino acid sequence YDASNRAT (SEQ ID NO: 72); and CDR3 represented by amino acid sequence CQQRSNWPLFT (SEQ ID NO: 73). Those skilled in the art know the allowable variation of the CDRs of the light chain while maintaining their contribution to binding of PSMA. For example, a human 2C6PSMA binding domain can include a light chain variable region comprising a CDR1, which CDR1 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR1 amino acid sequence set forth in SEQ ID No. 71. For example, a human 2C6PSMA binding domain can include a light chain variable region comprising a CDR2, which CDR2 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR2 amino acid sequence set forth in SEQ ID No. 72. For example, a human 2C6PSMA binding domain can include a light chain variable region comprising a CDR3, which CDR3 includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the CDR3 amino acid sequence set forth in SEQ ID No. 73. In one embodiment, the human 2C6PSMA binding domain comprises a light chain variable region comprising three of the foregoing light chain variable region CDRs.

Transmembrane domain

A CAR of the invention (e.g., a PSMA-CAR) can include a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the subject CARs is a region capable of spanning the plasma membrane of a cell (e.g., an immune cell or a precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of the CAR.

In some embodiments, the transmembrane domain is naturally associated with one or more domains in the CAR. In some embodiments, the transmembrane domains may be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

The transmembrane domain may be derived from natural or synthetic sources. When the source is native, the domain may be derived from any membrane bound protein or transmembrane protein, e.g., a type I transmembrane protein. When the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into the cell membrane, e.g., an artificial hydrophobic sequence. Examples of transmembrane domains of particular utility in the present invention include, but are not limited to, transmembrane domains derived from (i.e., including at least the transmembrane region(s) of): the α, β or ζ chain of a T cell receptor, CD28, CD3, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134(OX-40), CD137(4-1BB), CD154(CD40L), Toll-like receptor 1(TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR 9. In some embodiments, the transmembrane domain may be synthetic, in which it will predominantly comprise hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.

The transmembrane domain described herein can bind to any antigen binding domain described herein, any intracellular domain, or any other domain described herein that can be included in the subject CAR.

In some embodiments, the transmembrane domain further comprises a hinge region. The subject CAR of the invention may further comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, the domain promotes proper protein folding of the CAR. The hinge region is an optional component for the CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, artificial hinges made from polypeptides that can be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG 4).

In some embodiments, the subject CARs of the present disclosure include a hinge region that connects the antigen binding domain to the transmembrane domain, which in turn is connected to the intracellular domain. The hinge region is preferably capable of supporting an antigen binding domain to recognize and bind to an antigen on a target cell (see, e.g., Hudecek et al, Cancer Immunol. Res. (2015)3(2): 125-. In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure that optimally recognizes the specific structure and density of a target antigen on a cell (such as a tumor cell) (Hudecek et al, supra). The flexibility of the hinge region allows the hinge region to adopt a number of different conformations.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a hinge region derived from CD 8).

The hinge region may be about 4 amino acids to about 50 amino acids in length, e.g., about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

Suitable hinge regions can be readily selected and can possess any number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

For example, the hinge region includes glycine polymer (G) n, glycine-serine polymers (including, e.g., (GS) n, (GSGGS) n (SEQ ID NO:1), and (GGGS) n (SEQ ID NO:2), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used; both Gly and Ser are relatively unstructured and can therefore be used as neutral adhesion (teter) between ingredients. Glycine polymers may be used; glycine enters significantly even more than alanine

Space, and is less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. computational. chem. (1992)2: 73-142). Exemplary hinge regions may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO:4), GGSGG (SEQ ID NO:5), GSGSGSG (SEQ ID NO:6), GSGGG (SEQ ID NO:7), GGGSG (SEQ ID NO:8), GSSSG (SEQ ID NO:9), and the like.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al, Proc.Natl.Acad.Sci.USA (1990)87(1) 162-; and Huck et al, Nucleic Acids Res. (1986)14(4): 1779-1789. As a non-limiting example, the immunoglobulin hinge region may comprise one of the following amino acid sequences: DKKHT (SEQ ID NO: 74); CPPC (SEQ ID NO: 75); CPEPKSCDTPPPCPR (SEQ ID NO:76) (see, e.g., Glaser et al, J.biol.chem. (2005)280: 41494-41503); ELKTPLGDTTHT (SEQ ID NO: 77); KSCDKTHTCP (SEQ ID NO: 78); KCCVDCP (SEQ ID NO: 79); KYGPPCP (SEQ ID NO: 80); EPKSCDKTHTCPPCP (SEQ ID NO:81) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:82) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:83) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:84) (human IgG4 hinge); and the like.

The hinge region may comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. In one embodiment, the hinge region may comprise one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the hinge of human IgG1 can be substituted with Tyr such that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 85); see, e.g., Yan et al, j.biol.chem. (2012)287: 5891-. In one embodiment, the hinge region may comprise an amino acid sequence derived from human CD8 or a variant thereof.

The transmembrane domain may be associated with any hinge region and/or may include one or more of the transmembrane domains described herein. In one embodiment, the transmembrane domain comprises a CD8 transmembrane domain. In one embodiment, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain. In some embodiments, the subject CAR comprises a CD8 hinge region having the amino acid sequence set forth below:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:86), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO:87)。

those skilled in the art are aware of the allowable variations of the transmembrane domain while maintaining its intended function. For example, in some embodiments, a subject CAR of the invention includes a transmembrane domain comprising a CD8 hinge region, the CD8 hinge region including an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 86. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region, the CD8 hinge region comprising the amino acid sequence set forth in SEQ ID NO: 86.

In some embodiments, the subject CARs of the invention comprise a transmembrane domain comprising a CD8 hinge region, which CD8 hinge region is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence depicted in SEQ ID No. 87. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region encoded by the nucleic acid sequence set forth in SEQ ID NO: 87.

In some embodiments, the subject CAR comprises a CD8 hinge region having the amino acid sequence set forth below: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:88),

it may be encoded by the nucleic acid sequences set forth below:

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO:89)。

those skilled in the art are aware of the allowable variations of the transmembrane domain while maintaining its intended function. For example, in some embodiments, a subject CAR of the invention comprises a transmembrane domain comprising a CD8 transmembrane domain, the CD8 transmembrane domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 88. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 transmembrane domain, the CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 88.

In some embodiments, the subject CARs of the invention comprise a transmembrane domain comprising a CD8 transmembrane domain, which CD8 transmembrane domain is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 89. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 transmembrane domain, the CD8 transmembrane domain encoded by the nucleic acid sequence set forth in SEQ ID No. 89.

In some embodiments, the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain having the amino acid sequences set forth below:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:90)，

it may be encoded by the nucleic acid sequences set forth below:

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO:91)。

those skilled in the art are aware of the allowable variations of the transmembrane domain while maintaining its intended function. For example, in some embodiments, the subject CARs of the invention comprise a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, the CD8 hinge region and CD8 transmembrane domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 90. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, the CD8 hinge region and CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID No. 90.

In some embodiments, the subject CARs of the invention comprise a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, the CD8 hinge region and CD8 transmembrane domain being encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 91. In one embodiment, the CAR comprises a transmembrane domain comprising a CD8 hinge region and a CD8 transmembrane domain, which CD8 hinge region and CD8 transmembrane domain are encoded by the nucleic acid sequence set forth in SEQ ID NO: 91.

A spacer domain may be incorporated between the extracellular domain and the transmembrane domain of the CAR or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or an intracellular domain in a polypeptide chain. The spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain may be a short oligopeptide or polypeptide linker, e.g., between 2 and 10 amino acids in length. For example, a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR.

Intracellular signaling domains

The subject CARs of the invention also include an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activating at least one of the effector functions of the CAR-expressing cell (e.g., an immune cell). Intracellular signaling domains transduce effector function signals and direct cells (e.g., immune cells) to perform their specialized functions, such as injuring and/or destroying target cells.

Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, costimulatory molecules, and any molecule that acts synergistically to initiate signal transduction in T cells, as well as any derivative or variant of these elements and any synthetic sequence with the same functional capacity.

Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor complex or any homolog thereof, e.g., the eta, FcsRI gamma and beta chains, MB 1(Iga) chain, B29(Ig) chain, etc., the human CD3 zeta chain, CD3 polypeptides (delta, and), Syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD 28. In one embodiment, the intracellular signaling domain may be the zeta chain of human CD3, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor bearing an Immunoreceptor Tyrosine Activation Motif (ITAM), and combinations thereof.

In one embodiment, the intracellular signaling domain of the CAR comprises any portion of one or more co-stimulatory molecules, such as at least one signaling domain from: CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

Other examples of intracellular domains include fragments or domains from one or more molecules or receptors including, but not limited to: TCR, CD3 ζ, CD3 γ, CD3, CD3, CD86, ordinary FcRy, FcRb (Fcib), CD79a, CD79B, Fc γ Rlla, DAP10, DAP 12, T Cell Receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family protein, lymphocyte function-related antigen-1 (LFA-1), CD2, LIGHT, NKG 22, B2-H2, ligand binding specifically to CD2, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), AMF 2, NKp 2 (KLRF 2), CD127, CD 36160, CD2, VLITβ, GAI-72, GAI-X2, GAITGA-2, GAITGA 2, GAITX 2, GAITGA 2, GAITX 2, GAIT, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD 96(Tactile), CEACAM1, CRT AM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-6), SLAM (SLAMF 6, CD150, IPO-3), BLAME (SLAMF 6), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/bp, NKp 6, NKG 26, Toll-like receptor 1(TLR 6), TLR 72, TLR6, a synthetic molecule of any other co-stimulatory molecule having the same sequence or combination of the same stimulatory capacity as any of the synthetic molecule described herein.

Additional examples of intracellular domains include, but are not limited to, several types of intracellular signaling domains of various other immune signaling receptors, including, but not limited to, first, second and third generation T cell signaling proteins, including CD3, B7 family costimulators, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, j.clin.oncol. (2015)33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, front. immunol. (2015)6:195), signaling domains such as NKp30 (B7-H6) (see, e.g., Zhang et al., j.immunol. (2012)189(5):2290-2299), and DAP 12 (see, e.g., Topfer et al., j.immunol. (2015)194(7):3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3 z.

Intracellular signaling domains suitable for use in the subject CARs of the invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by a cell; changes in transcription of a target gene; changes in protein activity; changes in cellular behavior (e.g., cell death); cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response; etc.) in response to activation of the CAR (i.e., activation by an antigen and a dimerizing agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif described below. In some embodiments, the intracellular signaling domain comprises a DAP10/CD 28-type signaling strand. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but diffuses in the cytoplasm.

Intracellular signaling domains suitable for use in the subject CARs of the invention include intracellular signaling polypeptides comprising the Immunoreceptor Tyrosine Activation Motif (ITAM). In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of the subject CAR comprises 3 ITAM motifs.

In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor that contains an Immunoreceptor Tyrosine Activation Motif (ITAM), such as, but not limited to, Fc γ RI, Fc γ RIIA, Fc γ RIIC, Fc γ RIIIA, FcRL5 (see, e.g., Gillis et al, front.

Suitable intracellular signaling domains may be ITAM motif-containing portions derived from ITAM motif-containing polypeptides. For example, a suitable intracellular signaling domain may be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc receptor I γ chain), CD3D (CD3), CD3E (CD3), CD3G (CD3 γ), CD3Z (CD3 ζ), and CD79A (antigen receptor complex associated protein α chain).

In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-related protein; TYRO protein tyrosine kinase binding protein; killing activating receptor-related protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc receptor Igamma chain; Fc receptor gamma-chain; Fc-RI-gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 chain (also known as CD 3D; CD 3-delta; T3D; CD3 antigen, subunit; CD 3; CD3d antigen, polypeptide (TiT3 complex); OKT3 chain; T-cell receptor T3 chain; T-cell surface glycoprotein CD3 chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 chain (also known as CD3e, T-cell surface antigen T3/Leu-4 chain, T-cell surface glycoprotein CD3 chain, AI504783, CD3, CD3, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-Z, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also referred to as B-cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane bound immunoglobulin associated protein; surface IgM associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in the FN3 CARs of the present disclosure comprises a DAP10/CD 28-type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in the FN3 CARs of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR ζ, FcR γ, FcR β, CD3 γ, CD3, CD3, CD5, CD22, CD79a, CD79b, or CD66 d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 ζ.

While it is generally possible to employ the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. With respect to the use of truncated portions of intracellular signaling domains, such truncated portions can be used in place of the entire chain, so long as they transduce effector functional signals. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

The intracellular signaling domain described herein can bind to any antigen binding domain described herein, any transmembrane structure described herein, or any other domain described herein that can be included in a CAR.

In one embodiment, the intracellular domain of the subject CAR comprises a 4-1BB domain comprising the amino acid sequence set forth below:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:92), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:93)，

or by a nucleic acid sequence as set forth in:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:94)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a 4-1BB domain, the 4-1BB domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 92. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain, the 4-1BB domain comprising the amino acid sequence set forth in SEQ ID NO: 92.

In some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a 4-1BB domain, the 4-1BB domain being encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a nucleic acid sequence recited in SEQ ID No. 93 or 94. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain encoded by a nucleic acid sequence set forth in SEQ ID NO 93 or 94.

In one embodiment, the intracellular domain of the subject CAR comprises an ICOS domain comprising the amino acid sequence set forth below:

TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL(SEQ ID NO:203)，

it may be encoded by the nucleic acid sequences set forth below:

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA(SEQ ID NO:204)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising an ICOS domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 203. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence set forth in SEQ ID NO. 203.

In some embodiments, a subject CAR of the invention comprises an intracellular domain comprising an ICOS domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID No. 204. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 204.

In one embodiment, the intracellular domain of the subject CAR comprises a variant ICOS domain comprising the amino acid sequence set forth below:

TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTL(SEQ ID NO:95)，

it may be encoded by the nucleic acid sequences set forth below:

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTA(SEQ ID NO:96)。

variant ICOS domains are also referred to herein as ICOS (ymnm).

One skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising an ICOS domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 95. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain comprising the amino acid sequence set forth in SEQ ID NO: 95.

In some embodiments, a subject CAR of the invention comprises an intracellular domain comprising an ICOS domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 96. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 96.

In one embodiment, the intracellular domain of the subject CAR comprises the CD3 zeta domain comprising the amino acid sequence set forth in seq id no:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:97)，

it may be encoded by the nucleic acid sequences set forth below:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:98)，

or by a nucleic acid sequence as set forth in:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:99)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 97. In one embodiment, the CAR comprises an intracellular domain comprising the zeta domain of CD3 comprising the amino acid sequence depicted in SEQ ID NO: 97.

In some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain being encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a nucleic acid sequence recited in SEQ ID No. 98 or 99. In one embodiment, the CAR comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain being encoded by a nucleic acid sequence set forth in SEQ ID NO 98 or 99.

The CD3 zeta domain may comprise the amino acid sequence set forth in:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:100)，

it may be encoded by the nucleic acid sequences set forth below:

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:101)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 100. In one embodiment, the subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain, which CD3 zeta domain comprises the amino acid sequence depicted in SEQ ID NO: 100.

In some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain, the CD3 zeta domain being encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 101. In one embodiment, the subject CAR of the invention comprises an intracellular domain comprising a CD3 zeta domain encoded by the nucleic acid sequence depicted in SEQ ID NO 101.

In one embodiment, the CAR comprises an intracellular domain comprising the CD3 zeta domain of the amino acid sequence set forth in SEQ ID NO 97 or 100.

In an exemplary embodiment, the intracellular domain of the subject CAR comprises the 4-1BB domain and CD3 zeta domain comprising the amino acid sequences recited below:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:102), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:103)，

Or by a nucleic acid sequence as set forth in:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:104)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 102. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and the CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 102.

In some embodiments, the subject CARs of the invention comprise an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and CD3 zeta domain being encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 103 or 104. In one embodiment, the CAR comprises an intracellular domain comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and CD3 zeta domain being encoded by the nucleic acid sequences recited in SEQ ID NO 103 or 104.

In an exemplary embodiment, the intracellular domain of the subject CAR comprises an ICOS domain and a CD3 zeta domain comprising the amino acid sequence set forth below:

TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:205)，

it may be encoded by the nucleic acid sequences set forth below:

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:206)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, the ICOS domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 205. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain, the ICOS domain and the CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 205.

In some embodiments, the subject CARs of the invention comprise an intracellular domain comprising an ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 206. In one embodiment, the CAR comprises an intracellular domain comprising an ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 206.

In an exemplary embodiment, the intracellular domain of the subject CAR comprises a variant ICOS domain and a CD3 zeta domain comprising the amino acid sequence set forth below:

TKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:207)，

it may be encoded by the nucleic acid sequences set forth below:

ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:208)。

one skilled in the art will know the allowable variations of the intracellular domains, but retain their intended function. For example, in some embodiments, a subject CAR of the invention comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 207. In one embodiment, the CAR comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 207.

In some embodiments, the subject CARs of the invention comprise an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 208. In one embodiment, the CAR comprises an intracellular domain comprising a variant ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 208.

CAR sequences

The subject CAR of the invention can be selected from the group consisting of J591 murine PSMA-CAR, humanized J591 PSMA-CAR, 1C3 human PSMA-CAR, 2a10 human PSMA-CAR, 2F5 human PSMA-CAR, and 2C6 human PSMA-CAR.

In one embodiment, the subject CAR of the invention is a J591 murine PSMA-CAR. In one embodiment, the J591 murine PSMA-CAR comprises the amino acid sequence set forth below:

MALPVTALLLPLALLLHAARPGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKGGGGSGGGGSSGGGSEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSASSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:105)，

It may be encoded by the nucleic acid sequences set forth below:

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGCGCTAGCTCCGGAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:106)。

in one embodiment, a subject CAR of the invention is a humanized PSMA-CAR, e.g., a humanized J591 PSMA-CAR. In such embodiments, the humanized PSMA-CAR comprises any of the heavy and light chain variable regions disclosed in PCT publication nos. WO2017212250a1 and WO2018033749a 1. For example, humanized PSMA-CARs of the invention can include scfvs comprising any of the heavy and light chain variable regions disclosed therein, see, e.g., the sequences recited in table 19 of the disclosure.

In one embodiment, the subject CAR of the invention is a 1C3 human PSMA-CAR. In one embodiment, the 1C3 human PSMA-CAR comprises the amino acid sequence set forth in:

MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAVPWGSRYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKSGKAPKLLIFDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:107)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:108)。

in one embodiment, the subject CAR of the invention is a 2a10 human PSMA-CAR. In one embodiment, 2a10 human PSMA-CAR comprises the amino acid sequence set forth in:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQTGFLWSSDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGYGSGTDFTLTINSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:109)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:110)。

in one embodiment, the subject CAR of the invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and the CD3 zeta domain comprising the amino acid sequences recited below:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:111)，

It may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:112)。

in one embodiment, the subject CAR of the invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises an ICOS domain and a CD3 zeta domain, the ICOS domain and CD3 zeta domain comprising the amino acid sequence recited below:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:209)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:210)。

in one embodiment, the subject CAR of the invention is a 2F5 human PSMA-CAR. In one embodiment, the 2F5 human PSMA-CAR comprises a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and the CD3 zeta domain comprising the amino acid sequence recited below:

MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSNWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWNSLKASDTAMYYCARQTGFLWSFDLWGRGTLVTVSSGGGGSGGGGSGGGGSAIQLTQSPSSLSASVGDRVTITCRASQDISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMNMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:211)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:212)。

in one embodiment, the subject CAR of the invention is a 2C6 human PSMA-CAR. In one embodiment, the 2C6 human PSMA-CAR comprises the amino acid sequence set forth in:

MALPVTALLLPLALLLHAARPEVQLVQSGSEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCASPGYTSSWTSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:113)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:114)。

one skilled in the art is aware of the permissible variations in the sequence of the subject CAR while maintaining its functionality.

For example, in some embodiments, a subject CAR of the invention is a J591 murine PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 105. In one embodiment, the CAR is a J591 murine PSMA-CAR, the J591 murine PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 105.

For example, in some embodiments, the subject CAR of the invention is a humanized J591 PSMA-CAR. The humanized J591PSMA-CAR comprises a humanized J591PSMA binding domain comprising heavy and light chain variable regions selected from any of the heavy and light chain variable region sequences recited in table 19. In some embodiments, a humanized J591PSMA-CAR comprises a 4-1BB domain and a CD3 zeta domain.

For example, in some embodiments, a subject CAR of the invention is a 1C3 human PSMA-CAR, the 1C3 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to an amino acid sequence recited in SEQ ID No. 107. In one embodiment, the CAR is a 1C3 human PSMA-CAR, the 1C3 human PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 107.

For example, in some embodiments, a subject CAR of the invention is a 2a10 human PSMA-CAR, the 2a10 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 109. In one embodiment, the CAR is a 2A10 human PSMA-CAR, the 2A10 human PSMA-CAR comprising the amino acid sequence recited in SEQ ID NO: 109.

For example, in some embodiments, the subject CAR of the invention is a 2F5 human PSMA-CAR. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 111. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and the CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 111. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain, the ICOS domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 209. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO: 209. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 211. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain, the variant ICOS domain and CD3 zeta domain comprising the amino acid sequence set forth in SEQ ID NO 211. For example, in some embodiments, a subject CAR of the invention is a 2C6 human PSMA-CAR comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence recited in SEQ ID No. 113. In one embodiment, the CAR is a 2C6 human PSMA-CAR comprising the amino acid sequence set forth in SEQ ID NO: 113.

In some embodiments, a subject CAR of the invention is a J591 murine PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 106. In one embodiment, the CAR is a J591 murine PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 106.

For example, in some embodiments, a subject CAR of the invention is a 1C3 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 108. In one embodiment, the CAR is a 1C3 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 108. For example, in some embodiments, a subject CAR of the invention is a 2a10 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 110. In one embodiment, the CAR is a 2A10 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 110. For example, in some embodiments, the subject CAR of the invention is a 2F5 human PSMA-CAR. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 112. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a 4-1BB domain and a CD3 zeta domain, the 4-1BB domain and the CD3 zeta domain being encoded by the nucleic acid sequence recited in SEQ ID NO: 112. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 210. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising an ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence set forth in SEQ ID NO: 210. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 212. In one embodiment, the CAR is a 2F5 human PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain encoded by the nucleic acid sequence recited in SEQ ID No. 212. For example, in some embodiments, a subject CAR of the invention is a 2C6 human PSMA-CAR encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in SEQ ID No. 114. In one embodiment, the CAR is a 2C6 human PSMA-CAR encoded by the nucleic acid sequence set forth in SEQ ID NO: 114.

In certain embodiments, a subject CAR of the invention can comprise any of the amino acid sequences corresponding to SEQ ID NO 209, 211, or 227 and 236.

Accordingly, the present invention provides modified immune cells or their precursor cells, such as modified T cells, comprising a Chimeric Antigen Receptor (CAR) having affinity for prostate membrane antigen (PSMA) on a target cell (e.g., a prostate cancer cell). In some embodiments, the CAR comprises a PSMA-binding domain. In some embodiments, the CAR comprises a murine PSMA binding domain. In one embodiment, the CAR comprises a J591 murine PSMA binding domain. In one embodiment, the CAR comprises a humanized J591 PSMA binding domain. In some embodiments, the CAR comprises a human PSMA-binding domain. In some embodiments, the CAR comprises a human PSMA-binding domain selected from the group consisting of: 1C3, 2a10, 2F5, and 2C6 human PSMA binding domains.

Thus, the subject CAR of the invention comprises a PSMA-binding domain and a transmembrane domain. In one embodiment, the CAR comprises a PSMA-binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region. In one embodiment, the CAR comprises a PSMA-binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 transmembrane domain. In one embodiment, the CAR comprises a PSMA-binding domain and a transmembrane domain, wherein the transmembrane domain comprises a CD8 hinge region and a CD8 transmembrane domain.

Thus, the subject CARs of the invention comprise a PSMA-binding domain, a transmembrane domain, and an intracellular domain. In one embodiment, the CAR comprises a PSMA-binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a 4-1BB domain. In one embodiment, the CAR comprises a PSMA binding domain, a transmembrane domain, and an endodomain, wherein the endodomain comprises a CD3 zeta domain. In one embodiment, the CAR comprises a PSMA binding domain, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a 4-1BB domain and a CD3 zeta domain.

C. Dominant negative receptor and switch receptor

The present invention provides compositions and methods of modified immune cells or precursors thereof, such as modified T cells, that include a dominant negative receptor and/or a switch receptor. Thus, in some embodiments, the immune cell has been genetically modified to express a dominant negative receptor and/or a switch receptor. As used herein, the term "dominant negative receptor" refers to a molecule designed to reduce the effect of a negative signal transduction molecule (e.g., the effect of a negative signal transduction molecule on a modified immune cell of the present invention). The dominant negative receptors of the invention can utilize an extracellular domain associated with a negative signal to bind to and reduce the effect of a negative signaling molecule, such as TGF- β or PD-1. Such dominant negative receptors are described herein. For example, a modified immune cell comprising a dominant negative receptor can bind to a negative signaling molecule in the microenvironment of the modified immune cell and reduce the effect that the negative signaling molecule may have on the modified immune cell.

In addition to reducing the effects of negative signal transduction molecules, the switch receptors of the present invention may also be designed to convert negative signals to positive signals using the inclusion of an intracellular domain associated with positive signals. Described herein are switch receptors designed to convert a negative signal to a positive signal. Thus, the switch receptor includes an extracellular domain associated with a negative signal and/or an intracellular domain associated with a positive signal.

Tumor cells create an immunosuppressive microenvironment that serves to protect the tumor cells from immune recognition and elimination. This immunosuppressive microenvironment may limit the effectiveness of immunosuppressive therapies, such as CAR-T cell therapy. The secreted cytokine transforming growth factor beta (TGF β) directly inhibits the function of cytotoxic T cells and additionally induces the formation of regulatory T cells to further suppress the immune response. In the context of prostate cancer, T cell immunosuppression due to TGF β has been previously described (Donkor et al, 2011; Shalapour et al, 2015). To reduce the immunosuppressive effects of TGF- β, immune cells may be modified to express a dominant negative receptor that is a dominant negative receptor for TGF- β.

In some embodiments, the dominant negative receptor is a truncated variant of the wild-type protein that is associated with a negative signal. In some embodiments, the dominant negative receptor is a dominant negative receptor for TGF- β. Thus, in some embodiments, the dominant negative receptor for TGF- β is a truncated variant of the wild-type TGF- β receptor. In some embodiments, the dominant negative receptor is a truncated dominant negative variant of a TGF-beta type II receptor (TGF-beta RII-DN). In one embodiment, the TGF β RII-DN comprises the amino acid sequence recited below:

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSSG(SEQ ID NO:115)，

It may be encoded by the nucleic acid sequences set forth below:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGA(SEQ ID NO:116)。

those skilled in the art are aware of the allowable variations in the sequence of TGF β RII-DN while maintaining its intended function. For example, in some embodiments, a dominant negative receptor of the invention is a TGF- β RII-DN including an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 115. In one embodiment, the dominant negative receptor is a TGF- β RII-DN including the amino acid sequence set forth in SEQ ID NO: 115.

In some embodiments, a dominant negative receptor of the invention is a TGF- β RII-DN encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a nucleic acid sequence recited in SEQ ID NO. 116. In one embodiment, the dominant negative receptor is a TGF- β RII-DN encoded by the nucleic acid sequence set forth in SEQ ID NO: 116.

In one embodiment, a switch receptor suitable for use in the present invention is the PD1-CTM-CD28 receptor. When expressed in cells, the PD1-CTM-CD28 receptor converts the negative PD1 signal to a positive CD28 signal. The PD1-CTM-CD28 receptor includes variants of the PD1 extracellular domain, the CD28 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-CTM-CD28 receptor comprises the amino acid sequence set forth in:

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:117)，

it may be encoded by the nucleic acid sequences set forth below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:118)。

one of skill in the art is aware of the permissible changes to the PD1-CTM-CD28 receptor while maintaining its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in a cell). Thus, the PD1-CTM-CD28 receptor of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-CTM-CD28 receptor amino acid sequence set forth in SEQ ID No. 117. Thus, the PD1-CTM-CD28 receptor of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-CTM-CD28 receptor nucleic acid sequence as set forth in SEQ ID No. 118.

In one embodiment, a switch receptor suitable for use in the present invention is the PD1-PTM-CD28 receptor. When expressed in cells, the PD1-PTM-CD28 receptor converts the negative PD1 signal to a positive CD28 signal. The PD1-PTM-CD28 receptor includes variants of the PD1 extracellular domain, the PD1 transmembrane domain, and the CD28 cytoplasmic domain. In one embodiment, the PD1-PTM-CD28 receptor comprises the amino acid sequence set forth in:

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:119)，

it may be encoded by the nucleic acid sequences set forth below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:120)。

one skilled in the art is aware of the permissible changes in PD1-PTM-CD28 receptor while maintaining its intended biological activity (e.g., converting a negative PD1 signal to a positive CD28 signal when expressed in a cell). Thus, the PD1-PTM-CD28 receptor of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-PTM-CD28 receptor amino acid sequence set forth in SEQ ID NO 119. Thus, the PD1-PTM-CD28 receptor of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-PTM-CD28 receptor nucleic acid sequence set forth in SEQ ID NO 120.

In one embodiment, a transducible receptor suitable for use in the present invention is PD1^A132LThe PTM-CD28 receptor. When expressed in cells, PD1^A132LThe PTM-CD28 receptor converts the negative PD1 signal into a positive CD28 signal. It was found that the point mutation of PD1 at amino acid position 132 (alanine substituted with leucine) (A132L) increased its affinity for PD-L1 by a factor of two (see, e.g., Zhang et al, Immunity (2004)20(3), 337-347). PD1^A132LThe PTM-CD28 receptor comprises variants of the PD1 extracellular domain, the PD1 transmembrane domain, and the CD28 cytoplasmic domain with an amino acid substitution at position 132 (a 132L). In one embodiment, the PD1^A132LThe PTM-CD28 receptor comprises the amino acid sequence as set out below: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:121),

it may be encoded by the nucleic acid sequences set forth below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGC(SEQ ID NO:122)。

one skilled in the art is aware of PD1^A132LPermissive changes of the PTM-CD28 receptor while maintaining its intended biological activity (e.g. conversion of negative PD1 signal to positive CD28 signal when expressed in cells). Thus, the PD1 of the present invention ^A132LThe PTM-CD28 receptor may comprise an amino acid sequence corresponding to PD1 as depicted in SEQ ID NO:121^A132L-the PTM-CD28 receptor amino acid sequence has a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. Thus, the PD1 of the present invention^A132LThe PTM-CD28 receptor may be encoded by a nucleic acid sequence corresponding to PD1 as depicted in SEQ ID NO. 122^A132L-the PTM-CD28 receptor nucleic acid sequence has a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.

In one embodiment, a switch receptor suitable for use in the present invention is the PD1-4-1BB receptor. When expressed in a cell, the PD1-4-1BB receptor (also referred to herein as PD1-BB) converts the negative PD1 signal to a positive 4-1BB signal. In one embodiment, the PD1-4-1BB receptor comprises the amino acid sequence set forth below

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:213)，

It may be encoded by the nucleic acid sequences set forth below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:214)。

one of skill in the art is aware of the permissible changes to the PD1-4-1BB receptor while maintaining its intended biological activity (e.g., converting negative PD1 signaling to positive 4-1BB signaling when expressed in a cell). Thus, a receptor of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-4-1BB receptor amino acid sequence set forth in SEQ ID No. 213. Thus, the PD1-4-1BB receptor of the present invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the PD1-4-1BB receptor nucleic acid sequence depicted in SEQ ID NO 214.

In one embodiment, a transducible receptor suitable for use in the present invention is PD1^A132LThe 4-1BB receptor. When expressed in cells, PD1^A132LThe-4-1 BB receptor (also referred to herein as PD1 × BB) converts the negative PD1 signal to a positive 4-1BB signal. In one embodiment, the PD1^A132LThe-4-1 BB receptor comprises the amino acid sequence as set forth in: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKLQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVIYIWAPLAGTCGVLLLSLVITLYCKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:215),

it may be encoded by the nucleic acid sequences set forth below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:216)。

one skilled in the art is aware of PD1^A132L-tolerance of the 4-1BB receptor while maintaining its intended biological activity (e.g., negative PD1 is believed to be expressed in cellsThe number transitions to a positive 4-1BB signal). Thus, the PD1 of the present invention^A132LThe-4-1 BB receptor may comprise an amino acid sequence corresponding to PD1 as depicted in SEQ ID NO:215^A132L-the 4-1BB receptor amino acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. Thus, the PD1 of the present invention ^A132LThe 4-1BB receptor may be encoded by a nucleic acid sequence which is identical to PD1 as depicted in SEQ ID NO 216^A132L-the 4-1BB receptor nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.

In one embodiment, a suitable transducible receptor for use in the present invention is the TGF β R-IL12R β 1 receptor. When expressed in cells, the TGF β R-IL12R β 1 receptor converts negative TGF- β signaling to positive IL-12 signaling. In one embodiment, the TGF β R-IL12R β 1 receptor comprises the amino acid sequence set forth in seq id no:

MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISLMLMVYIRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM(SEQ ID NO:123)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGAGGCGGCGGTCGCTGCTCCGCGTCCCCGGCTGCTCCTCCTCGTGCTGGCGGCGGCGGCGGCGGCGGCGGCGGCGCTGCTCCCGGGGGCGACGGCGTTACAGTGTTTCTGCCACCTCTGTACAAAAGACAATTTTACTTGTGTGACAGATGGGCTCTGCTTTGTCTCTGTCACAGAGACCACAGACAAAGTTATACACAACAGCATGTGTATAGCTGAAATTGACTTAATTCCTCGAGATAGGCCGTTTGTATGTGCACCCTCTTCAAAAACTGGGTCTGTGACTACAACATATTGCTGCAATCAGGACCATTGCAATAAAATAGAACTTCCAACTACTGTAAAGTCATCACCTGGCCTTGGTCCTGTGGAACTGGCAGCTGTCATTGCTGGACCAGTGTGCTTCGTCTGCATCTCACTCATGTTGATGGTCTATATCAGGGCCGCACGGCACCTGTGCCCGCCGCTGCCCACACCCTGTGCCAGCTCCGCCATTGAGTTCCCTGGAGGGAAGGAGACTTGGCAGTGGATCAACCCAGTGGACTTCCAGGAAGAGGCATCCCTGCAGGAGGCCCTGGTGGTAGAGATGTCCTGGGACAAAGGCGAGAGGACTGAGCCTCTCGAGAAGACAGAGCTACCTGAGGGTGCCCCTGAGCTGGCCCTGGATACAGAGTTGTCCTTGGAGGATGGAGACAGGTGCAAGGCCAAGATG(SEQ ID NO:124)。

one of skill in the art is aware of the allowable changes to the TGF β R-IL12R β 1 receptor while maintaining its intended biological activity (e.g., converting negative TGF- β signaling to positive IL-12 signaling when expressed in a cell). Thus, the TGF-beta R-IL12R beta 1 receptor of the invention may include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the TGF-beta R-IL12R beta 1 receptor amino acid sequence set forth in SEQ ID NO 123. Thus, a TGF-beta R-IL12R beta 1 receptor of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF-beta R-IL12R beta 1 receptor nucleic acid sequence set forth in SEQ ID NO 124.

In one embodiment, a suitable transducible receptor for use in the present invention is the TGF β R-IL12R β 2 receptor. When expressed in cells, the TGF β R-IL12R β 2 receptor converts negative TGF- β signaling to positive IL-12 signaling. In one embodiment, the TGF β R-IL12R β 2 receptor comprises the amino acid sequence set forth in seq id no:

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML (SEQ ID NO:125), which may be encoded by the nucleic acid sequence set forth in SEQ ID NO:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACCAGCAAAAGGTGTTTGTTCTCCTAGCAGCCCTCAGACCTCAGTGGTGTAGCAGAGAAATTCCAGATCCAGCAAATAGCACTTGCGCTAAGAAATATCCCATTGCAGAGGAGAAGACACAGCTGCCCTTGGACAGGCTCCTGATAGACTGGCCCACGCCTGAAGATCCTGAACCGCTGGTCATCAGTGAAGTCCTTCATCAAGTGACCCCAGTTTTCAGACATCCCCCCTGCTCCAACTGGCCACAAAGGGAAAAAGGAATCCAAGGTCATCAGGCCTCTGAGAAAGACATGATGCACAGTGCCTCAAGCCCACCACCTCCAAGAGCTCTCCAAGCTGAGAGCAGACAACTGGTGGATCTGTACAAGGTGCTGGAGAGCAGGGGCTCCGACCCAAAGCCAGAAAACCCAGCCTGTCCCTGGACGGTGCTCCCAGCAGGTGACCTTCCCACCCATGATGGCTACTTACCCTCCAACATAGATGACCTCCCCTCACATGAGGCACCTCTCGCTGACTCTCTGGAAGAACTGGAGCCTCAGCACATCTCCCTTTCTGTTTTCCCCTCAAGTTCTCTTCACCCACTCACCTTCTCCTGTGGTGATAAGCTGACTCTGGATCAGTTAAAGATGAGGTGTGACTCCCTCATGCTC(SEQ ID NO:126)。

one of skill in the art is aware of the allowable changes to the TGF β R-IL12R β 2 receptor while maintaining its intended biological activity (e.g., converting negative TGF- β signaling to positive IL-12 signaling when expressed in a cell). Thus, the TGF-beta R-IL12R beta 2 receptor of the invention may include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the TGF-beta R-IL12R beta 2 receptor amino acid sequence set forth in SEQ ID NO 125. Thus, a TGF-beta R-IL12R beta 2 receptor of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF-beta R-IL12R beta 2 receptor nucleic acid sequence set forth in SEQ ID NO 126.

In one embodiment, a switch receptor suitable for use in the present invention is the TIM3-CD28 receptor. When expressed in cells, the TIM3-CD28 receptor converts the negative TIM-3 signal to a positive CD28 signal. In one embodiment, the TIM3-CD28 receptor includes the amino acid sequence set forth below: MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:127),

it may be encoded by the nucleic acid sequences set forth below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:128)。

one of skill in the art is aware of the permissible changes to the TIM3-CD28 receptor while maintaining its intended biological activity (e.g., converting a negative TIM-3 signal to a positive CD28 signal when expressed in a cell). Thus, the TIM3-CD28 receptor of the present invention may include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the TIM3-CD28 receptor amino acid sequence set forth in SEQ ID No. 127. Thus, the TIM3-CD28 receptor of the present invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the TIM3-CD28 receptor nucleic acid sequence set forth in SEQ ID No. 128.

Other suitable dominant negative receptors and switch receptors for use in the present invention are described in PCT publication WO2013019615A2, the disclosure of which is incorporated herein by reference.

D. Bispecific antibodies

The present invention provides compositions and methods of modified immune cells or precursors thereof, such as modified T cells, comprising nucleic acids encoding bispecific antibodies. Thus, in some embodiments, the immune cell has been genetically modified to express the bispecific antibody. As used herein, "bispecific antibody" refers to an antibody having binding specificity for at least two different epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods of making bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced using co-expression of two immunoglobulin heavy/light chain pairs. See, e.g., Milstein et al (1983) Nature 305: 537-39. Alternatively, bispecific antibodies can be made using chemical linkage. See, e.g., Brennan et al (1985) Science 229: 81. Bispecific antibodies include bispecific antibody fragments. See, e.g., Holliger et al (1993) Proc. Natl. Acad. Sci. U.S.A.90:6444-48, Gruber et al (1994) J.Immunol.152: 5368.

In certain embodiments, the modified cells of the invention include a CAR and a bispecific antibody having affinity for prostate membrane antigen (PSMA) on a target cell. In certain embodiments, the modified cells of the invention secrete bispecific antibodies.

In one embodiment, a bispecific antibody comprises a first antigen-binding domain that binds a first antigen and a second antigen-binding domain that binds a second antigen. In some embodiments, a bispecific antibody comprises an antigen binding domain comprising first and second single chain variable fragment (scFv) molecules. In one embodiment, the first and second antigen binding domains bind to an antigen on a target cell and an antigen on an activated T cell.

In one embodiment, the bispecific antibody comprises specificity for at least one antigen on an activated T cell. An activated T cell antigen includes an antigen found on the surface of a T cell that can activate another cell. The activated T cell antigen may bind to a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which are required for efficient response of lymphocytes to antigens. Examples of activated T cell antigens may include, but are not limited to, CD3, CD4, CD8, T Cell Receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, or a fragment thereof. In some embodiments, the bispecific antibody comprises specificity for the T cell antigen CD 28.

Other co-stimulatory elements are also within the scope of the invention. In these examples, the bispecific antibody recognizes a T cell antigen and may be referred to as a bispecific T cell adaptor (BiTE). However, the present invention is not limited to the use of any particular bispecific antibody. Rather, any bispecific antibody or BiTE can be used. Bispecific antibodies or BiTE molecules can also be expressed as soluble proteins specific for at least one target cell-associated antigen.

In one embodiment, the bispecific antibody comprises more than one antigen binding domain. In this embodiment, the at least one antigen binding domain comprises a synthetic antibody, a human antibody, a humanized antibody, a single chain variable fragment, a single domain antibody, an antigen binding fragment thereof, and any combination thereof. Techniques for making human and humanized antibodies are described elsewhere herein.

In some embodiments, the bispecific antibody comprises more than one antigen binding domain, wherein at least one antigen binding domain binds a negative signaling molecule (e.g., a negative signaling molecule that can be found in the microenvironment of the bispecific antibody-secreting cell) or an interaction partner thereof (e.g., a receptor). In some embodiments, at least one antigen binding domain of a bispecific antibody binds TGF- β or an interaction partner (e.g., receptor) thereof. In some embodiments, at least one antigen binding domain of the bispecific antibody binds PD-1 or an interaction partner thereof. In one embodiment, at least one antigen binding domain of the bispecific antibody binds TGF- β R. In another embodiment, at least one antigen binding domain of the bispecific antibody binds PD-L1.

In some embodiments, the bispecific antibody comprises at least one antigen binding domain that binds to a molecule on a T cell and activates the T cell. For example, bispecific antibodies of the invention may include a superagonist anti-CD 28 binding domain as described in U.S. patent 7,585,960, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the bispecific antibody comprises at least one antigen binding domain that binds PD-L1. For example, bispecific antibodies of the present disclosure may include, but are not limited to, PD-L1 binding domains derived from 10a5, 13G4, or 1B12 as described in PCT publication WO2007005874a2, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a bispecific antibody comprises at least one antigen binding domain that binds to a TGF- β receptor, e.g., TGF β RII. For example, bispecific antibodies of the present disclosure may include, but are not limited to, TGF β RII binding domains derived from TGF1 or TGF3 disclosed in U.S. patent No. 8,147,834, the contents of which are incorporated herein by reference in their entirety.

Thus, in one embodiment, a bispecific antibody of the present disclosure comprises at least one antigen binding domain that binds PD-L1 or TGF β RII and an antigen binding domain that binds CD 28.

In some embodiments, the target cell antigen may be the same antigen to which the T cell receptor binds or may be a different antigen. Target cell antigens include any Tumor Associated Antigen (TAA) or viral, bacterial and parasitic antigens, or any fragment thereof. The target cell antigen may include any type of ligand that defines a target cell. For example, a target cell antigen can be selected to recognize a ligand that serves as a cellular marker on a target cell associated with a particular disease state. Thus, the cell markers can serve as ligands for the antigen binding domain in bispecific antibodies, including those associated with viral, bacterial and parasitic infections, autoimmune diseases and cancer cells.

In some embodiments, the target cell antigen is the same antigen as the activated T cell antigen, including but not limited to CD3, CD4, CD8, T Cell Receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and fragments thereof. In one aspect, the invention includes a nucleic acid encoding a bispecific antibody that includes a bispecific to an antigen on a target cell and an antigen on an activating T cell, wherein the T cell transiently secretes the bispecific antibody. Techniques for engineering and expressing bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305:537(1983), WO 93/08829, and Traunecker et al, EMBO J.10:3655(1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be made using the following techniques: production of antibody Fc heterodimer molecules by engineered electrostatic steering (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al, Science 229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.148(5):1547-1553 (1992)); the "diabody" technique was used to make bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (scFv) dimers (see, e.g., Gruber et al, J.Immunol,152:5368 (1994)); and trispecific antibodies were prepared as described in Tutt et al j. immunol.147:60 (1991). Engineered antibodies having three or more functional antigen binding sites (including "Octopus antibodies") (see, e.g., US 2006/0025576a1) are also included herein. Bispecific antibodies can be constructed by linking two different antibodies or portions thereof. For example, a bispecific antibody can include a Fab, F (ab ')2, Fab', scFv, and sdAb from two different antibodies.

Bispecific antibodies of the invention include bispecific antibodies having affinity for PD-L1 and CD 28. In one embodiment, the 13G4-1211PD-L1/CD28 bispecific antibody of the invention comprises an amino acid sequence set forth in: MGWSCIILFLVATATGVHSAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI (SEQ ID NO:129),

it may be encoded by the nucleic acid sequences set forth below:

ATGGGGTGGTCGTGTATCATCCTGTTCCTGGTCGCGACAGCAACCGGCGTGCATTCGGCCATACAGCTGACCCAGAGCCCCTCCTCCCTCTCCGCTTCCGTGGGGGACCGCGTGACAATCACGTGCCGCGCCAGCCAGGGAATCTCCTCGGCCCTCGCCTGGTACCAGCAGAAACCCGGGAAGGCTCCCAAGCTGCTCATCTACGATGCCTCCTCGCTTGAGTCGGGCGTGCCATCCAGGTTCTCCGGATCCGGGTCCGGAACCGACTTTACACTCACGATTTCCTCTCTGCAGCCCGAGGACTTCGCCACATACTACTGTCAGCAGTTCAACTCCTACCCATTCACCTTCGGCCCGGGCACCAAGGTGGACATCAAGTCTGGCGGGGGAGGCTCCGAAGTCCAGCTCGTGGAATCCGGGGGCGGTCTCGTGCAGCCAGGCCGGAGTCTGCGCCTGTCTTGCGCTGCCTCGGGGATCACTTTCGACGACTACGGCATGCATTGGGTTCGCCAGGCCCCAGGGAAGGGGTTGGAGTGGGTCAGTGGCATTTCATGGAACAGGGGGCGCATCGAATACGCCGACTCCGTTAAGGGCAGATTCACCATCTCGCGCGATAACGCCAAAAACAGTCTCTACCTCCAGATGAACTCGCTTCGAGCAGAGGATACTGCCCTGTACTATTGCGCGAAGGGACGCTTCCGCTACTTTGACTGGTTTCTGGACTACTGGGGCCAGGGGACACTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGAT(SEQ ID NO:130)。

one of skill in the art is aware of the permissible variations of 13G4-1211PD-L1/CD28 bispecific antibodies while maintaining their intended biological activity (e.g., binding to PD-L1 and CD 28). Thus, the 13G4-1211PD-L1/CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the 13G4-1211PD-L1/CD28 bispecific antibody amino acid sequence recited in SEQ ID NO. 129. Thus, a 13G4-1211PD-L1/CD28 bispecific antibody of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the 13G4-1211PD-L1/CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO 130.

Bispecific antibodies of the invention include bispecific antibodies having affinity for PD-L1 and CD 28. In one embodiment, the 10a5-1412PD-L1/CD28 bispecific antibody of the present invention comprises the amino acid sequence set forth in: MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI (SEQ ID NO:131),

it may be encoded by the nucleic acid sequences set forth below:

ATGGGCTGGAGTTGCATCATTCTCTTCCTCGTGGCGACCGCAACAGGGGTGCACTCCGACATCCAGATGACCCAGTCCCCGAGTTCCCTGTCTGCTTCCGTGGGAGATCGCGTGACTATCACCTGCCGGGCTTCCCAGGGCATCTCTTCCTGGCTGGCGTGGTACCAGCAGAAACCAGAAAAGGCTCCTAAGTCCCTGATCTACGCAGCTTCGTCCCTCCAATCCGGCGTCCCCTCTCGCTTCTCCGGCTCCGGATCCGGCACCGACTTCACGCTGACAATCTCGAGTTTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAACTCCTACCCTTACACCTTCGGCCAGGGCACAAAGCTCGAAATCAAGTCGGGGGGGGGCGGGTCGCAGGTCCAGCTGGTGCAGTCCGGCGCCGAAGTCAAGAAGCCCGGAGCAAGTGTGAAAGTGTCGTGCAAGGCAAGTGGGTATACCTTCACCTCATACGACGTACACTGGGTGCGCCAGGCGCCCGGTCAGCGCCTTGAGTGGATGGGCTGGCTCCACGCCGACACCGGCATTACCAAGTTCTCTCAGAAGTTCCAGGGAAGAGTGACCATAACACGCGACACCAGTGCTTCCACAGCTTACATGGAACTTTCGAGTCTGAGATCCGAGGACACAGCCGTGTATTACTGTGCCCGTGAGCGCATCCAGCTGTGGTTCGACTACTGGGGGCAGGGCACCCTCGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGAT(SEQ ID NO:132)。

one of skill in the art is aware of the permissible variations of the 10a5-1412PD-L1/CD28 bispecific antibody while maintaining its intended biological activity (e.g., binding to PD-L1 and CD 28). Thus, a 10a5-1412PD-L1/CD28 bispecific antibody of the invention can include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the 10a5-1412PD-L1/CD28 bispecific antibody amino acid sequence recited in SEQ ID No. 131. Thus, a 10A5-1412PD-L1/CD28 bispecific antibody of the invention can be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence 132 recited in SEQ ID NO: with the 10A5-1412PD-L1/CD28 bispecific antibody nucleic acid sequence recited in SEQ ID NO: 132.

Bispecific antibodies of the invention include bispecific antibodies having affinity for PD-L1 and CD 28. In one embodiment, the 1B12-1412PD-L1/CD28 bispecific antibody of the present invention comprises the amino acid sequence set forth in: MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKSGGGGSQVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTVTVSSGGSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEI (SEQ ID NO:133),

it may be encoded by the nucleic acid sequences set forth below:

ATGGGCTGGAGTTGCATCATCCTCTTTCTAGTCGCCACGGCCACCGGCGTACACTCAGAGATCGTGCTGACACAGTCGCCTGCGACGCTGTCGCTCAGTCCAGGGGAGCGCGCTACTCTCTCCTGCCGCGCGTCGCAGAGCGTGTCGTCCTACTTGGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCGCGCCTGCTGATATACGACGCCTCGAACAGAGCCACGGGCATCCCCGCCCGTTTTAGTGGCTCCGGGTCGGGGACCGACTTCACTCTGACAATCTCATCCCTCGAGCCCGAGGATTTCGCCGTGTACTACTGTCAGCAGCGCTCGAATTGGCCAACCTTCGGGCAGGGGACGAAAGTTGAGATCAAAAGCGGCGGCGGGGGCAGCCAGGTCCAGCTCGTCCAGTCTGGCGCCGAGGTCAAAAAGCCGGGCTCTTCGGTCAAGGTCTCCTGCAAGACTTCCGGCGACACCTTCTCCTCCTATGCTATCTCCTGGGTGCGGCAGGCCCCGGGGCAGGGCCTGGAGTGGATGGGAGGCATCATCCCAATCTTTGGGAGGGCCCACTACGCCCAGAAGTTCCAGGGACGCGTGACAATCACCGCAGACGAGTCCACATCCACTGCCTACATGGAGTTGTCCTCGCTCCGGTCGGAGGATACTGCCGTGTACTTCTGCGCCCGGAAGTTCCACTTCGTGTCAGGCTCCCCCTTCGGGATGGACGTGTGGGGACAAGGAACCGTGACGGTGTCGTCGGGGGGCTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGAT(SEQ ID NO:134)。

one of skill in the art is aware of the permissible variations of the 1B12-1412PD-L1/CD28 bispecific antibody while maintaining its intended biological activity (e.g., binding to PD-L1 and CD 28). Thus, a 1B12-1412PD-L1/CD28 bispecific antibody of the invention may comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the 1B12-1412PD-L1/CD28 bispecific antibody amino acid sequence recited in SEQ ID No. 133. Thus, a 1B12-1412PD-L1/CD28 bispecific antibody of the invention can be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the 1B12-1412PD-L1/CD28 bispecific antibody nucleic acid sequence recited in SEQ ID No. 134.

Bispecific antibodies of the invention include those having affinity for TGF-beta receptor type II (TGF-beta RII) and CD 28. In one embodiment, a TGF-beta R-1-1412 TGF-beta RII/CD28 bispecific antibody of the invention includes an amino acid sequence set forth in:

MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK(SEQ ID NO:135)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGGTTGGTCCTGCATCATCCTGTTTCTCGTGGCCACCGCCACCGGCGTGCACTCCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTCGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGTGGAGGGGGCGGTTCACAGCTGCAGGTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAACAGTTATTTCTCCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGAGTTTCTATTATGGTGAAAAAACCTACTACAACCCGTCCCTCAAGAGCCGAGCCACCATATCCATTGACACGTCCAAGAGCCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTCCGAGAGGGCCTACTATGATTCGGGGAGTTATAGACTCCTGGGGCCAGGGAACCCTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGATTAAG(SEQ ID NO:136)。

those skilled in the art are aware of the permissible variations of TGF-beta R-1-1412 TGF-beta RII/CD28 bispecific antibodies while retaining their intended biological activities (e.g., binding to TGF-beta RII and CD 28). Thus, a TGF- β R-1-1412 TGF- β RII/CD28 bispecific antibody of the invention may include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF- β R-1-1412 TGF- β RII/CD28 bispecific antibody amino acid sequence set forth in SEQ ID NO. 135. Thus, a TGF- β R-1-1412 TGF- β RII/CD28 bispecific antibody of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF- β R-1-1412 TGF- β RII/CD28 bispecific antibody nucleic acid sequence set forth in SEQ ID NO 136.

Bispecific antibodies of the invention include those having affinity for TGF-beta receptor type II (TGF-beta RII) and CD 28. In one embodiment, a TGF-beta R-3-1412 TGF-beta RII/CD28 bispecific antibody of the invention includes an amino acid sequence set forth in:

MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQSVRSFLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYSWGWIRQPPGKGLEWIGSFYYSGITYYSPSLKSRIIISEDTSKNQFSLKLSSVTAADTAVYYCASGFTMIRGALDYWGQGTLVTVSSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSSVEGGSGGSGGSGGSGGVMDDIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK(SEQ ID NO:137)，

it may be encoded by the nucleic acid sequences set forth below:

ATGGGTTGGTCCTGCATCATCCTGTTTCTCGTGGCCACCGCCACCGGCGTGCACTCCGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGAAGTTTCTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGTGGAGGGGGCGGTTCACAGCTACAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTATCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTCCTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGCCTGGAGTGGATTGGGAGTTTCTATTACAGTGGGATCACCTACTACAGCCCGTCCCTCAAGAGTCGAATTATCATATCCGAAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGCGGGTTTACTATGATTCGGGGAGCCCTTGACTACTGGGGCCAGGGAACCCTGGTGACGGTGTCGTCGGGGGGCGGGGGGAGTCAGGTGCAGCTGGTGCAGTCCGGAGCCGAGGTAAAGAAGCCAGGCGCTTCCGTCAAGGTGTCATGCAAGGCCTCAGGCTACACCTTCACAAGCTATTACATCCACTGGGTGCGCCAAGCTCCCGGTCAGGGCTTGGAGTGGATCGGGTGCATTTACCCAGGGAACGTCAACACAAACTACAACGAGAAGTTCAAGGATCGGGCAACCCTGACCGTGGACACATCCATCTCTACCGCCTACATGGAGCTGTCACGCCTGCGCTCTGATGACACCGCAGTGTACTTCTGTACCAGGAGTCACTACGGCCTGGACTGGAACTTTGATGTCTGGGGCCAGGGAACCACCGTGACGGTGTCCAGTGTGGAGGGCGGTAGTGGCGGCTCTGGTGGGTCCGGAGGCTCAGGCGGCGTGATGGATGACATTCAGATGACCCAGAGTCCCTCCTCCCTCTCCGCTTCCGTCGGAGACCGCGTGACCATCACTTGTCACGCCTCACAGAATATCTACGTGTGGCTGAACTGGTACCAACAGAAGCCCGGCAAGGCCCCCAAGCTGCTTATCTATAAAGCGTCCAACCTCCACACGGGAGTCCCTTCCCGCTTCTCCGGATCCGGCAGTGGGACGGACTTCACACTCACAATCTCGTCGCTGCAGCCAGAGGACTTTGCGACGTACTACTGCCAGCAGGGCCAGACCTACCCATATACTTTCGGCGGCGGGACCAAGGTGGAGATTAAG(SEQ ID NO:138)。

those skilled in the art are aware of the permissible variations of TGF-beta R-3-1412 TGF-beta RII/CD28 bispecific antibodies while retaining their intended biological activities (e.g., binding to TGF-beta RII and CD 28). Thus, a TGF- β R-3-1412 TGF- β RII/CD28 bispecific antibody of the invention may include an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF- β R-3-1412 TGF- β RII/CD28 bispecific antibody amino acid sequence set forth in SEQ ID NO: 137. Thus, a TGF- β R-3-1412 TGF- β RII/CD28 bispecific antibody of the invention may be encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a TGF- β R-3-1412 TGF- β RII/CD28 bispecific antibody nucleic acid sequence set forth in SEQ ID NO 138.

Other suitable bispecific antibodies for use in the present invention are described in PCT publication No. WO2016122738a1, the disclosure of which is incorporated herein by reference.

E. Nucleic acids and expression vectors

The invention provides nucleic acids encoding a CAR and/or a dominant negative receptor and/or a switch receptor. In one embodiment, a nucleic acid of the disclosure includes a nucleic acid sequence encoding a subject CAR of the invention (e.g., a PSMA-CAR). In one embodiment, a nucleic acid of the present disclosure includes a nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor (e.g., PD1-PTM-CD28 receptor).

In some embodiments, the nucleic acids of the disclosure provide for the production of a CAR and/or dominant negative receptor and/or switch receptor as described herein, as in a mammalian cell. In some embodiments, the nucleic acids of the present disclosure provide for amplification of nucleic acids encoding a CAR and/or a dominant negative receptor and/or a switch receptor.

As described herein, the subject CARs include an antigen binding domain, a transmembrane domain, and an intracellular domain. Accordingly, the disclosure provides nucleic acids encoding the antigen binding domain, transmembrane domain, and intracellular domain of the subject CARs. As described herein, various dominant negative receptors and switch receptors are provided. Accordingly, the present invention provides nucleic acids encoding dominant negative receptors and/or switch receptors.

In some embodiments, the nucleic acid encoding the CAR is isolated from a nucleic acid encoding a dominant negative receptor and/or a switch receptor. In an exemplary embodiment, the nucleic acid encoding the CAR and the nucleic acid encoding the dominant negative receptor and/or the switch receptor are present in the same nucleic acid.

In some embodiments, the nucleic acids of the invention include nucleic acids comprising a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence. In some embodiments, the nucleic acids of the invention include nucleic acids comprising a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence separated by a linker. The linkers used in the invention (e.g., in the context of linking the CAR coding sequence and the dominant negative receptor and/or transformation receptor coding sequence) allow for multiple proteins (e.g., polycistronic or bicistronic sequences) to be encoded by the same nucleic acid sequence, which are translated into multiple proteins that dissociate into separate protein components. For example, the linker used in a nucleic acid of the disclosure containing a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence allows a CAR and dominant negative receptor and/or switch receptor to be translated as a polyprotein that dissociates into individual CAR and dominant negative receptor and/or switch receptor components.

In some embodiments, the linker comprises a nucleic acid sequence encoding an Internal Ribosome Entry Site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that facilitates direct access of internal ribosomes to the initiation codon (such as ATG) of a protein coding region, thereby allowing cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRES obtainable from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular Endothelial Growth Factor (VEGF); fibroblast growth factor 2; an insulin-like growth factor; translation initiation factor eIF 4G; yeast transcription factors TFIID and HAP 4; and IRES obtainable from, for example, heart virus, rhinovirus, foot and mouth disease virus, HCV, Friedel's murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select an IRES suitable for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein, which is post-translationally cleaved into component proteins. The use of the term "self-cleavage" does not mean a hydrolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those skilled in the art, including, but not limited to, those found in members of the picornaviridae family, such as Foot and Mouth Disease Virus (FMDV), equine rhinitis A virus (ERAV0), Spodoptera litura virus (TaV), and porcine iron and wire archaea virus-1 (PTV-1), and cardioviruses such as Theileria virus (Theilovir) and encephalomyocarditis virus (encarditis virus). The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A", "E2A", "P2A", and "T2A", respectively. Those skilled in the art will be able to select a self-cleaving peptide suitable for use in the present invention.

In some embodiments, nucleic acids of the disclosure include nucleic acid sequences comprising a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence separated by a linker comprising a T2A peptide sequence. In some embodiments, the T2A peptide sequence includes amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO:139), which may be encoded by nucleic acid sequence GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO: 140). In some embodiments, a linker comprising a T2A peptide sequence may further comprise a spacer sequence described herein. For example, a linker comprising a T2A peptide sequence may further comprise a spacer comprising the amino acid sequence SGRSGGG (SEQ ID NO:141), which may be encoded by nucleic acid sequence TCCGGAAGATCTGGCGGCGGA (SEQ ID NO: 142).

In some embodiments, nucleic acids of the disclosure include nucleic acid sequences comprising a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence separated by a linker that includes an F2A peptide sequence. In some embodiments, the F2A peptide sequence includes amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:143), which may be encoded by nucleic acid sequence GTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG (SEQ ID NO: 144).

In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a universally expressed protease that is present in trans-homo-clan and processes its precursors prior to protein secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. The skilled artisan knows consensus recognition sequences (or "furin cleavage sites") for various furins, including, but not limited to, Arg-X-Lys-Arg (SEQ ID NO:145) or Arg-X-Arg-Arg (SEQ ID NO:146), and Arg-X-X-Arg (SEQ ID NO:147), such as Arg-Gln-Lys-Arg (SEQ ID NO:148), where X is any naturally occurring amino acid. Another example of a furin cleavage site is X1-Arg-X2-X3-Arg (SEQ ID NO:149), wherein X1 is Lys or Arg, X2 is any naturally occurring amino acid, and X3 is Lys or Arg. One skilled in the art will be able to select suitable furin cleavage sites for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, linkers comprising the nucleic acid sequences encoding furin and F2A, linkers comprising the nucleic acid sequences encoding furin and E2A, linkers comprising the nucleic acid sequences encoding furin and P2A, linkers comprising the nucleic acid sequences encoding furin and T2A. One skilled in the art will be able to select suitable combinations for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin and the 2A peptide. Various spacer sequences are known in the art, including, but not limited to, Glycine Serine (GS) spacers such as (GS) n, (GSGGS) n (SEQ ID NO:1), and (GGGS) n (SEQ ID NO:2), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences including, but not limited to: GGSG (SEQ ID NO:4), GGSGG (SEQ ID NO:5), GSGSG (SEQ ID NO:6), GSGGG (SEQ ID NO:7), GGGSG (SEQ ID NO:8), GSSSG (SEQ ID NO:9), and the like. One skilled in the art will be able to select suitable spacer sequences for use in the present invention.

In some embodiments, the nucleic acids of the disclosure include a nucleic acid sequence comprising a CAR coding sequence and a dominant negative receptor and/or switch receptor coding sequence separated by a furin- (G4S)2-T2A (F-GS2-T2A) linker. The F-GS2-T2A linker may be encoded by the nucleic acid sequence CGTGCGAAGAGGGGCGGCGGGGGCTCCGGCGGGGGAGGCAGTGAGGGCCGCGGCTCCCTGCTGACCTGCGGAGATGTAGAAGAGAACCCAGGCCCC (SEQ ID NO:150) and may include the amino acid sequence RAKRGGGGSGGGGSEGRGSLLTCGDVEENPGP (SEQ ID NO: 151). One skilled in the art will recognize that the linkers of the present invention may include allowable sequence variations.

In some embodiments, the invention provides nucleic acids comprising nucleic acid sequences encoding the dominant negative receptors and/or switch receptors described herein. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor and a nucleic acid sequence encoding a CAR (e.g., PSMA-CAR) as described herein. In one embodiment, the nucleic acid sequence encoding the dominant negative receptor and/or the switch receptor and the nucleic acid sequence encoding the CAR are present on separate nucleic acids. In one embodiment, the nucleic acid sequence encoding the dominant negative receptor and/or the switch receptor and the nucleic acid sequence encoding the CAR are present within the same nucleic acid. In such embodiments, the nucleic acid sequence encoding the dominant negative receptor and/or the switch receptor and the nucleic acid sequence encoding the CAR are separated by a linker as described herein.

For example, a nucleic acid of the disclosure can include a nucleic acid sequence encoding a dominant receptor, a linker, and encoding a CAR. In one embodiment, the linker comprises a nucleic acid sequence encoding a 2A peptide (e.g., T2A). In exemplary embodiments, the nucleic acids of the disclosure can include a nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor and a nucleic acid sequence encoding a CAR separated by a linker that includes a nucleic acid sequence encoding T2A.

Thus, in one embodiment, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor, a nucleic acid sequence encoding a linker, and a nucleic acid sequence encoding a CAR. In one embodiment, the nucleic acids of the present disclosure include, from 5 'to 3', a nucleic acid sequence encoding a CAR, a CAR encoding a linker, a nucleic acid sequence encoding a dominant negative receptor, and/or a switch receptor.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the dominant negative receptor is TGF β RII-DN. In one embodiment, the CAR is a murine J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding murine J591 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding a TGF β RII-DN, a nucleic acid sequence comprising a T2A linker, and a nucleic acid encoding a murine J591 PSMA-CAR include the nucleic acid sequences recited below: ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCCAGACCTGGATCTGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATCTGTAAGGCCAGTCAAGATGTGGGTACTGCTGTAGACTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTACTAACGTTCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAAGGAGGCGGAGGATCTGGCGGCGGAGGAAGTTCTGGCGGAGGCAGCGAGGTGCAGCTGCAGCAGAGCGGACCCGAGCTCGTGAAGCCTGGAACAAGCGTGCGGATCAGCTGCAAGACCAGCGGCTACACCTTCACCGAGTACACCATCCACTGGGTCAAGCAGTCCCACGGCAAGAGCCTGGAGTGGATCGGCAATATCAACCCCAACAACGGCGGCACCACCTACAACCAGAAGTTCGAGGACAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGCGGAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGTGCCGCCGGTTGGAACTTCGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGCGCTAGCTCCGGAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 152).

In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid sequence encoding a humanized J591 PSMA-CAR. In one embodiment, the nucleic acids of the present disclosure comprise, from 5 'to 3': a nucleic acid encoding a humanized PSMA-CAR, a nucleic acid encoding a linker comprising a 2A peptide (e.g., T2A), and a nucleic acid encoding a dominant negative receptor and/or a switch receptor.

In one embodiment, the CAR is a humanized J591 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a humanized J591 PSMA-CAR. In one embodiment, the nucleic acid comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding a humanized J591 PSMA-CAR.

Humanized PSMA-CARs may include any of the heavy and light chain variable regions disclosed in PCT publication nos. WO2017212250a1 and WO2018033749a 1. For example, a humanized PSMA-CAR of the invention may comprise an scFv comprising any of the heavy and light chain variable regions disclosed herein. In some embodiments, the humanized J591 PSMA-CAR comprises a humanized J591PSMA binding domain comprising heavy and light chain variable regions selected from any of the heavy and light chain variable region sequences recited in table 19.

In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding human 1C3 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TGF β RII-DN, the nucleic acid sequence encoding the linker comprising T2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 1C3 PSMA-CAR include the nucleic acid sequences recited below:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:153)。

in one embodiment, the CAR is a human 2a10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding human 2a10 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TGF β RII-DN, the nucleic acid sequence encoding the linker comprising T2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2a10 PSMA-CAR include the nucleic acid sequences recited below:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:154)。

in one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TGF β RII-DN, the nucleic acid sequence encoding the linker comprising T2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2F5 PSMA-CAR include the nucleic acid sequences recited below:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:155)。

In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding a TGF β RII-DN, a nucleic acid sequence encoding a linker comprising T2A, and a nucleic acid sequence encoding human 2C6 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TGF β RII-DN, the nucleic acid sequence encoding the linker comprising T2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2C6 PSMA-CAR include the nucleic acid sequences recited below:

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:156)。

those skilled in the art are aware of the permissible variations of the nucleic acid sequences encoding TGF- β RII-DN and PSMA-CAR. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NO 152-156. In one embodiment, the nucleic acid sequence encoding TGF β RII-DN and murine J591 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 152. In one embodiment, the nucleic acid sequence encoding TGF β RII-DN and human 1C3 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 153. In one embodiment, the nucleic acid sequence encoding TGF β RII-DN and human 2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 154. In one embodiment, the nucleic acid sequence encoding TGF β RII-DN and human 2F5 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 155. In one embodiment, the nucleic acid sequence encoding TGF β RII-DN and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 156.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 1C3 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of PD1-CTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 1C3PSMA-CAR include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:157)。

in some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 2a10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2a10 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: the nucleic acid sequence encoding PD1-CTM-CD28, the nucleic acid sequence encoding a linker comprising F2A, and the nucleic acid sequence encoding human 2a10 PSMA-CAR include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:158)。

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of PD1-CTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2F5PSMA-CAR include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:159)。

in some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2C6 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of PD1-CTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2C6 PSMA-CAR include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:160)。

Those skilled in the art are aware of the permissible variations of the nucleic acid sequences encoding PD1-CTM-CD28 and PSMA-CAR. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NO 157-160. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 1C3 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 157. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2A10 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 158. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 159. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2C6 PSMA-CAR comprises the nucleic acid sequence set forth in SEQ ID NO: 160.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 1C3 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acids encoding the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 1C3 PSMA-CAR comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:161)。

in some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 2a10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1 ^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2a10 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acids encoding the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2a10 PSMA-CAR comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:162)。

in some embodimentsWherein, the nucleic acids of the present disclosure comprise, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acids encoding the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2F5 PSMA-CAR comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:163)。

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2C6 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acids encoding the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2C6 PSMA-CAR comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:164)。

those skilled in the art are aware of the code PD1^A132LTolerable variations of the nucleic acid sequences of PTM-CD28 and PSMA-CAR. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NO 161-164. In one embodiment, the encoded PD1 ^A132LThe nucleic acid sequence of PTM-CD28 and human 1C3 PSMA-CAR comprises the nucleic acid sequence depicted in SEQ ID NO: 161. In one embodiment, the encoded PD1^A132LThe nucleic acid sequences of PTM-CD28 and human 2A10 PSMA-CAR comprise the nucleic acid sequence depicted in SEQ ID NO: 162. In one embodiment, the encoded PD1^A132LThe nucleic acid sequences of PTM-CD28 and human 2F5 PSMA-CAR comprise the nucleic acid sequence depicted in SEQ ID NO. 163. In one embodiment, the encoded PD1^A132LThe nucleic acid sequences of PTM-CD28 and human 2C6 PSMA-CAR comprise the nucleic acid sequence depicted in SEQ ID NO: 164.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 1C3 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a linker including F2A, and nucleic acid sequences encoding human 1C3 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, the nucleic acid sequence encoding the linker including F2A, and the nucleic acid sequence encoding human 1C3 PSMA-CAR include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:165)。

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2a10 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a linker including F2A, and nucleic acid sequences encoding human 2a10 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2a10PSMA-CAR include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:166)。

in some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a linker including F2A, and nucleic acid sequences encoding human 2F5 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2F5 PSMA-CAR include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:167)。

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2C6 PSMA-CAR. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a linker including F2A, and nucleic acid sequences encoding human 2C6 PSMA-CAR. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2C6 PSMA-CAR include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGACGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGACGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:168)。

one skilled in the art knows the allowable variations of nucleic acid sequences encoding TIM3-CD28 and PSMA-CAR. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence recited in any one of SEQ ID NO 165-168. In one embodiment, the nucleic acid sequences encoding TIM3-CD28 and human 1C3 PSMA-CAR comprise the nucleic acid sequence set forth in SEQ ID NO: 165. In one embodiment, the nucleic acid sequences encoding TIM3-CD28 and human 2A10PSMA-CAR comprise the nucleic acid sequence set forth in SEQ ID NO: 166. In one embodiment, the nucleic acid sequences encoding TIM3-CD28 and human 2F5 PSMA-CAR comprise the nucleic acid sequence set forth in SEQ ID NO: 167. In one embodiment, the nucleic acid sequences encoding TIM3-CD28 and human 2C6 PSMA-CAR comprise the nucleic acid sequence set forth in SEQ ID NO: 168.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of PD1-CTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:217)。

the skilled artisan is aware of the permissible variations of the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 217. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID NO. 217.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1-CTM-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding PD1-CTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of PD1-CTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:218)。

the skilled artisan is aware of the permissible variations of nucleic acid sequences encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR including a variant ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 218. In one embodiment, the nucleic acid sequence encoding PD1-CTM-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID No. 218.

In some embodiments, the nucleic acids of the disclosure are coated from 5' to 3Comprises the following steps: a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132L-the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid encoding the nucleic acid sequence of human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:219)。

those skilled in the art are aware of the code PD1^A132L-permissible variation of the nucleic acid sequence of PTM-CD2 and 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO. 219. In one embodiment, the encoded PD1 ^A132LThe nucleic acid sequence of PTM-CD28 and human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence depicted in SEQ ID NO: 219.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodimentWherein the transducible receptor is PD1^A132LPTM-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of PTM-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132L-the nucleic acid sequence of PTM-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid encoding the nucleic acid sequence of human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:220)。

those skilled in the art are aware of the code PD1^A132LPermissive variation of nucleic acid sequence of PTM-CD28 and human 2F5 PSMA-CAR including variant ICOS domain and CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 220. In one embodiment, the encoded PD1 ^A132LThe nucleic acid sequence of PTM-CD28 and human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence depicted in SEQ ID NO: 220.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132L-4-1 BB. In one embodiment, the CAR is a device comprising an ICOS junctionHuman 2F5PSMA-CAR of domain and CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of 4-1BB, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LThe nucleic acid sequence of-4-1 BB, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the nucleic acid sequence of human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:221)。

those skilled in the art are aware of the code PD1^A132L-permissible variation in nucleic acid sequence of 4-1BB and human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 221. In one embodiment, the encoded PD1 ^A132LThe nucleic acid sequence of-4-1 BB and human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence depicted in SEQ ID NO 221.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is PD1^A132L-4-1 BB. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3':encoding PD1^A132L-a nucleic acid sequence of 4-1BB, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LThe nucleic acid sequence of-4-1 BB, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid encoding the nucleic acid sequence of human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprise the nucleic acid sequences recited below: ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACAC CGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:222)。

Those skilled in the art are aware of the code PD1^A132L-allowed variations in nucleic acid sequence of 4-1BB and human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 222. In one embodiment, the encoded PD1^A132LThe nucleic acid sequence of-4-1 BB and human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID NO 222.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, encoding the linker comprising F2A, and encoding the nucleic acid sequence of human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:223)。

One skilled in the art knows the allowable variations of the nucleic acid sequence encoding TIM3-CD28 and human 2F5PSMA-CAR including the ICOS domain and CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 223. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 2F5PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID NO: 223.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a switch receptor, a nucleic acid sequence encoding a linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a linker comprising F2A, and nucleic acid sequences encoding human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: nucleic acids encoding the nucleic acid sequence of TIM3-CD28, the nucleic acid sequence encoding the linker comprising F2A, and the nucleic acid sequence encoding the human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain include the nucleic acid sequences recited below:

ATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:224)。

One skilled in the art knows the allowable variations of nucleic acid sequences encoding TIM3-CD28 and human 2F5 PSMA-CAR including a variant ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 224. In one embodiment, the nucleic acid sequence encoding TIM3-CD28 and human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID No. 224.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a first switch receptor, a nucleic acid sequence encoding a first linker comprising F2A, a nucleic acid sequence encoding a second switch receptor, a nucleic acid sequence encoding a second linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the first switch receptor is TIM3-CD28 and the second switch receptor is PD1 ^A132L-4-1 BB. In one embodiment, the first switch receptor is PD1^A132L-4-1BB, and the second switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, the first and second connectors are identical. In one embodiment, the first and second connectors are different. Thus, in exemplary embodiments, the present inventionThe nucleic acids of the invention comprise, from 5 'to 3': encoding PD1^A132L-a nucleic acid sequence of 4-1BB, a nucleic acid sequence encoding a first linker comprising F2A, a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a second linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acid sequence of-4-1 BB, nucleic acid sequence encoding a first linker comprising F2A, nucleic acid sequence encoding TIM3-CD28, nucleic acid sequence encoding a second linker comprising F2A, and nucleic acid sequence encoding human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprise the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGTGAAGCAGACGTTGAACTTCGATTTGCTCAAACTTGCCGGTGACGTGGAATCCAATCCGGGGCCGATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:225)。

those skilled in the art are aware of the code PD1^A132L-4-1BB, TIM3-CD28, and human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60% of the nucleic acid sequence set forth in SEQ ID NO:225 At least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. In one embodiment, the encoded PD1^A132LThe nucleic acid sequence of-4-1 BB, TIM3-CD28, and human 2F5 PSMA-CAR comprising an ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID NO: 225.

In some embodiments, a nucleic acid of the present disclosure comprises, from 5 'to 3': a nucleic acid sequence encoding a first switch receptor, a nucleic acid sequence encoding a first linker comprising F2A, a nucleic acid sequence encoding a second switch receptor, a nucleic acid sequence encoding a second linker comprising F2A, and a nucleic acid sequence encoding a CAR. In one embodiment, the first switch receptor is TIM3-CD28 and the second switch receptor is PD1^A132L-4-1 BB. In one embodiment, the first switch receptor is PD1^A132L-4-1BB, and the second switch receptor is TIM3-CD 28. In one embodiment, the CAR is a human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, the first and second connectors are identical. In one embodiment, the first and second connectors are different. Thus, in an exemplary embodiment, a nucleic acid of the invention comprises, from 5 'to 3': encoding PD1 ^A132L-a nucleic acid sequence of 4-1BB, a nucleic acid sequence encoding a first linker comprising F2A, a nucleic acid sequence encoding TIM3-CD28, a nucleic acid sequence encoding a second linker comprising F2A, and a nucleic acid sequence encoding human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain. In one embodiment, from 5 'to 3' comprises: encoding PD1^A132LNucleic acid sequences of-4-1 BB, nucleic acid sequences encoding a first linker comprising F2A, nucleic acid sequences encoding TIM3-CD28, nucleic acid sequences encoding a second linker comprising F2A, and nucleic acid sequences encoding human 2F5 PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain include the nucleic acid sequences recited below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGGTGAAGCAGACGTTGAACTTCGATTTGCTCAAACTTGCCGGTGACGTGGAATCCAATCCGGGGCCGATGTTTTCACATCTTCCCTTTGACTGTGTCCTGCTGCTGCTGCTGCTACTACTTACAAGGTCCTCAGAAGTGGAATACAGAGCGGAGGTCGGTCAGAATGCCTATCTGCCCTGCTTCTACACCCCAGCCGCCCCAGGGAACCTCGTGCCCGTCTGCTGGGGCAAAGGAGCCTGTCCTGTGTTTGAATGTGGCAACGTGGTGCTCAGGACTGATGAAAGGGATGTGAATTATTGGACATCCAGATACTGGCTAAATGGGGATTTCCGCAAAGGAGATGTGTCCCTGACCATAGAGAATGTGACTCTAGCAGACAGTGGGATCTACTGCTGCCGAATCCAAATCCCAGGCATAATGAATGATGAAAAATTTAACCTGAAGTTGGTCATCAAACCAGCCAAGGTCACCCCTGCACCGACTCGGCAGAGAGACTTCACTGCAGCCTTTCCAAGGATGCTTACCACCAGGGGACATGGCCCAGCAGAGACACAGACACTGGGGAGCCTCCCTGACATAAATCTAACACAAATATCCACATTGGCCAATGAGTTACGGGACTCTAGGTTGGCCAATGACTTACGGGACTCCGGAGCAACCATCAGATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTACTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGAACATGAGAGCAGTGAACACAGCCAAAAAATCCAGACTCACAGATGTGACCCTAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:226)。

those skilled in the art are aware of the code PD1^A132L-allowed variations in nucleic acid sequence of 4-1BB, TIM3-CD28, and human 2F5 PSMA-CAR including a variant ICOS domain and a CD3 zeta domain. For example, in some embodiments, the nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO 226. In one embodiment, the encoded PD1 ^A132LThe nucleic acid sequence of-4-1 BB, TIM3-CD28, and human 2F5PSMA-CAR comprising a variant ICOS domain and a CD3 zeta domain comprises the nucleic acid sequence set forth in SEQ ID NO: 226.

In some embodiments, a nucleic acid of the present disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers, and the like. Suitable promoter and enhancer elements are known to those skilled in the art.

For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, λ P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoter; a herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; a promoter in the long terminal repeat of a retrovirus; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. Modifications of a reversible promoter derived from a first organism for use with a second organism (e.g., a first prokaryote and a second eukaryote, or a first eukaryote and a second prokaryote, etc.) are known in the art. Such reversible promoters, and systems based on such reversible promoters but also containing additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., the alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to the alcohol transactivator protein (A1cR), etc.), tetracycline regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., the rat glucocorticoid receptor promoter system, the human estrogen receptor promoter system, the retinoid promoter system, the thyroid promoter system, the ecdysone promoter system, the melasperidone (mifepristone) promoter system, etc.), metal regulated promoters (e.g., the metallothionein promoter system, etc.), related pathogen regulated promoters (e.g., the salicylic acid regulated promoter, the ethylene regulated promoter, the benzothiadiazole regulated promoter, etc.), (ii), Temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.), light regulated promoters, synthetic inducible promoters, etc.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, the CD4 gene promoter may be used; see, e.g., Salmon et al Proc. Natl. Acad. Sci. USA (1993)90: 7739; and Marodon et al (2003) Blood 101: 3416. As another example, the CD8 gene promoter may be used. NK cell-specific expression can be achieved by using NcrI (p46) promoter; see, e.g., Eckelhart et al blood (2011)117: 1565.

For expression in yeast cells, suitable promoters are constitutive promoters, such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, and the like; or a regulatable promoter such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for Pichia pastoris (Pichia)). Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, the bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operator promoter; hybrid promoters, e.g., lac/tac hybrid promoter, tac/trc hybrid promoter, trp/lac promoter, T7/lac promoter; a trc promoter; tac promoter, etc.; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. patent publication No. 20040131637), the pagC promoter (Pulkkien and Miller, J.Bacteriol. (1991)173(1): 86-93; Alpuche-Aranda et al, Proc.Natl.Acad.Sci.USA (1992)89(21):10079-83), the nirB promoter (Harborne et al.mol.Micro. (1992)6:2805-2813), etc. (see, e.g., Dunstan et al, Infect.Immun. (1999)67: 5133-5141; McKelvie et al., Vaccine (2004)22: 3243-3255; and Chatfield et al, Biotech. (1992)10: 888-892); a sigma 70 promoter, e.g., the consensus sigma 70 promoter (see, e.g., GenBank accession nos. AX79898, AX798961, and AX 798183); stationary phase promoters, e.g., dps promoter, spv promoter, etc.; a promoter derived from SPI-2 of the pathogenicity island (see, e.g., WO 96/17951); the actA promoter (see, e.g., Shetron-Rama et al, Impect. Immun. (2002)70: 1087-1096); the rpsM promoter (see, e.g., Valdivia and Falkow mol. Microbiol. (1996).22: 367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds.), Topics In Molecular and Structural Biology, protein- -Nucleic Acid interaction. Macmillan, London, UK, Vol.10, pp.143-162); the SP6 promoter (see, e.g., Melton et al, Nucl. acids Res. (1984)12: 7035); and the like. Suitable strong promoters for prokaryotes such as E.coli include, but are not limited to, Trc, Tac, T5, T7, and PLAmbda. Non-limiting examples of operators for bacterial host cells include the lactose promoter operator (the LacI repressor protein changes configuration when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (the TrpR repressor protein has a configuration that binds to the operator when complexed with tryptophan; the TrpR repressor protein has a configuration that does not bind to the operator in the absence of tryptophan), and the tac promoter operator (see, e.g., deBoer et al, Proc. Natl.Acad.Sci.U.S.A. (1983)80: 21-25).

Other examples of suitable promoters include the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the Mouse Mammary Tumor Virus (MMTV) or Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the epstein barr virus immediate early promoter, the rous sarcoma virus promoter, the EF-1 alpha promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence to which it is operably linked when expression is desired or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched by induction with an induction system. Suitable systems for inducing irreversible transformation are well known in the art, e.g., the induction of irreversible transformation can utilize Cre-lox mediated recombination (see, e.g., Fuhrmann-Benzakein, et al, proc.natl.acad.sci.usa (2000)28: e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art may be used to generate the irreversibly switched promoter. The methods, mechanisms, and requirements for site-specific recombination described elsewhere herein can be used to generate irreversibly switched promoters, and are well known in the art, see, e.g., Grindey et al, annual Review of Biochemistry (2006) 567-605; and tropip, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

In some embodiments, the nucleic acids of the disclosure further comprise a nucleic acid sequence encoding a TCR/CAR inducible expression cassette. In one embodiment, the TCR/CAR-inducible expression cassette is used to produce a transgenic polypeptide product that is released based on TCR/CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin biol. Ther. (2015)15(8): 1145-1154; and Abken, Immunotherapy (2015)7(5): 535-544. In some embodiments, the nucleic acids of the present disclosure further comprise a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter. In some embodiments, the cytokine operably linked to the T-cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

The nucleic acids of the present disclosure may be present in an expression vector and/or a cloning vector. Expression vectors may include selectable markers, origins of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for the production of the subject recombinant constructs. The following vectors may be provided by way of example, but should in no way be construed as limiting: bacteria: pBs, phage, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5(Pharmacia, Uppsala, Sweden). Eukaryotic organisms: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors typically have convenient restriction sites located near the promoter sequence to provide for insertion of a nucleic acid sequence encoding a heterologous protein. There may be a selectable marker operable in the expression host. Suitable expression vectors include, but are not limited to, viral vectors (e.g., poxvirus-based viral vectors; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994)35: 2543-2549; Borras et al., Gene Therr. (1999)6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995)92: 7700-7704; Sakamoto et al., H.Gene Ther. (1999)5: 1088-1097; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655; adeno-associated virus (see, e.g., Ali et al., Flann. Gene r. (1998)9:81-86, Acannery. 1997, Nat. 1997: 683690; Eur. J. Nat. 1997) 683690; Eur. J. Nat. Sci et al. (USA: 683690; Jorda.),691; Eur. J. (1999), rolling et al, hum. Gene Ther (1999)10: 641-648; ali et al, hum. mol. Genet. (1996)5: 591-594; srivastava in WO 93/09239, Samulski et al, J.Vir. (1989)63: 3822-3828; mendelson et al, Virol et al (1988)166: 154-165; and Flotte et al, Proc.Natl.Acad.Sci.USA (1993)90: 10613-10617); SV 40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al, Proc. Natl. Acad. Sci. USA (1997)94: 10319-23; Takahashi et al, J. Virol. (1999)73: 7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses, such as rous sarcoma disease, Harvey sarcoma virus, domestic leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and so on.

Additional expression vectors suitable for use are, for example, but not limited to, lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated viral vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid viral vectors, transposon mediated vectors, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al, 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Typically, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01/96584, WO01/29058, and U.S. Pat. No. 6,326,193).

In some embodiments, expression vectors (e.g., lentiviral vectors) can be used to introduce TCR/CARs and/or dominant negative receptors and/or switch receptors into immune cells or precursors thereof (e.g., T cells). Thus, an expression vector (e.g., a lentiviral vector) of the invention can include a nucleic acid encoding a TCR/CAR and/or a dominant negative receptor and/or a switch receptor. In some embodiments, the expression vector (e.g., a lentiviral vector) will include additional elements that facilitate functional expression of the TCR/CAR and/or dominant negative receptor and/or switch receptor encoded therein. In some embodiments, the expression vector comprising the nucleic acid encoding the TCR/CAR and/or the dominant negative receptor and/or the switch receptor further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1 alpha promoter). The use of the EF-1 a promoter can increase the efficiency of expression of downstream transgenes (e.g., nucleic acid sequences encoding TCR/CARs and/or dominant negative receptors and/or switch receptors). Physiological promoters (e.g., the EF-1. alpha. promoter) may be less likely to induce integration-mediated genotoxicity and may abolish the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and may be incorporated into the vectors of the invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises optional cis-acting sequences, which can increase titer and gene expression. One non-limiting example of an optional cis-acting sequence is the central polypurine tract (track) and central termination sequence (cPPT/CTS), which is important for efficient reverse transcription and nuclear import. Other optional cis-acting sequences are known to those of skill in the art and may be incorporated into the vectors of the invention (e.g., lentiviral vectors). In some embodiments, the vector further comprises a post-transcriptional regulatory element. Post-transcriptional regulatory elements can enhance RNA translation, enhance transgene expression, and stabilize RNA transcripts. An example of a post-transcriptional regulatory element is woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vectors of the invention further comprise a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vectors (e.g., lentiviral vectors) of the invention. The vectors of the invention may further include additional elements such as Rev Response Element (RRE) for RNA transport, packaging sequences, and 5 'and 3' Long Terminal Repeats (LTR). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, which includes the U3, R, and U5 regions. LTRs typically provide functions required for expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. In one embodiment, the vector of the invention (e.g., a lentiviral vector) comprises a LTR deleted for 3' U3. Thus, a vector of the invention (e.g., a lentiviral vector) can include any combination of elements described herein that enhance the efficiency of functional expression of a transgene. For example, in addition to nucleic acids encoding TCR/CARs and/or dominant negative receptors and/or switch receptors, vectors of the invention (e.g., lentiviral vectors) may also include WPRE sequences, cPPT sequences, RRE sequences, 5 ' LTRs, 3 ' U3 deleted LTRs '.

The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3' LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). Self-inactivating vectors can prevent viral transcription beyond the first round of viral replication. Thus, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be further delivered. Thus, self-inactivating vectors can greatly reduce the risk of producing replication-competent viruses.

In some embodiments, a nucleic acid of the invention can be an RNA, e.g., an RNA synthesized in vitro. Methods for in vitro synthesis of RNA are known to those skilled in the art; RNA including sequences encoding the disclosed TCR/CARs and/or dominant negative receptors and/or switch receptors can be synthesized using any known method. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al cancer Res (2010)15: 9053. Methods of introducing RNA into a host cell comprising a nucleotide sequence encoding a TCR/CAR and/or dominant negative receptor and/or switch receptor of the disclosure can be performed in vitro or ex vivo or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo with RNA comprising nucleotide sequences encoding the TCRs/CARs and/or dominant negative receptors and/or switch receptors of the disclosure.

To assess the expression of the polypeptide or portion thereof, the expression vector to be introduced into the cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected with the viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. The selectable marker and reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is not present or expressed in the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some easily detectable property (e.g., enzymatic activity). After the DNA is introduced into the recipient cells, the expression of the reporter gene is evaluated at an appropriate time. Suitable reporter genes may include, but are not limited to, the following genes: genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000 FEBS Letters 479: 79-82).

F. Modified immune cells

The invention provides modified immune cells or their precursor cells (e.g., T cells) that include a CAR and/or a dominant negative receptor and/or a switch receptor. Thus, such modified cells have specificity directed by the CAR expressed therein. For example, the modified cells of the invention comprising a PSMA-CAR are specific for PSMA on a target cell.

In some embodiments, the modified cell of the invention comprises a CAR. In one embodiment, the modified cell of the invention comprises a CAR having affinity for prostate membrane antigen (PSMA) on a target cell. In some embodiments, the modified cells of the invention comprise a dominant negative receptor and/or a switch receptor. In one embodiment, the modified cell of the invention comprises a dominant negative receptor capable of reducing the effect of a negative signal transduction molecule in a microenvironment. In one embodiment, the modified cells of the invention comprise a switch receptor capable of reducing the effect of negative signal transduction molecules in the microenvironment and converting negative signals to positive signals within the modified cells. In some embodiments, the modified cells of the invention comprise a CAR and a dominant negative receptor and/or a switch receptor. In one embodiment, the modified cell of the invention comprises a CAR having affinity for PSMA on a target cell and a dominant negative receptor and/or a switch receptor. The modified cells of the invention that include a dominant negative receptor and/or a switch receptor are capable of engaging a negative signaling molecule (e.g., an inhibitory ligand) in the microenvironment via their respective extracellular domains. In some embodiments, the modified cells of the invention comprising a dominant-negative receptor can reduce the effect of a negative signal transduction molecule in the microenvironment, wherein the dominant-negative receptor comprises an extracellular domain associated with a negative signal. In some embodiments, the modified cells of the invention comprising a switch receptor are capable of converting the action of a negative signal transduction molecule in a microenvironment into a positive signal, wherein the switch receptor comprises an extracellular domain associated with the negative signal and an intracellular domain associated with the positive signal.

In exemplary embodiments, the modified cells of the invention include a dominant negative receptor that is capable of reducing the effect of a negative signaling molecule. In one embodiment, the modified cell of the invention comprises a TGF β RII-DN.

In exemplary embodiments, the modified cells of the invention include a switch receptor that is capable of converting the action of a negative signal transduction molecule to a positive (e.g., activating) signal within the modified cell. In one embodiment, the modified cell of the invention comprises PD1-CTM-CD 28. In one embodiment, the modified cell of the invention comprises PD1^A132LPTM-CD 28. In one embodiment, the modified cell of the invention comprises TIM3-CD 28.

In an exemplary embodiment, the modified cell of the invention comprises a PSMA-CAR and a dominant negative receptor capable of reducing the effect of a negative signaling molecule. In one embodiment, the modified cells of the invention include murine J591PSMA-CAR and TGF β RII-DN. In one embodiment, the modified cells of the invention include a humanized J591PSMA-CAR and a TGF β RII-DN. In one embodiment, the modified cells of the invention include a human 1C3PSMA-CAR and a TGF β RII-DN. In one embodiment, the modified cells of the invention include a human 2a10PSMA-CAR and a TGF β RII-DN. In one embodiment, the modified cells of the invention include human 2F5PSMA-CAR and TGF β RII-DN. In one embodiment, the modified cells of the invention comprise a human 2C6PSMA-CAR and a TGF β RII-DN. Such modified cells (e.g., modified T cells), in addition to having affinity for PSMA on target cells, are also capable of reducing inhibitory TGF- β signaling from the microenvironment in which they are present.

In exemplary embodiments, the modified cells of the invention comprise a PSMA-CAR and an inhibitor capable of converting a negative signaling molecule within the modified cellAnd acts sexually on the transducible receptor for the positive signal. In one embodiment, the modified cells of the invention include murine J591 PSMA-CAR and PD1-CTM-CD 28. In one embodiment, the modified cell of the invention comprises a humanized J591 PSMA-CAR and PD1-PTM-CD 28. In one embodiment, the modified cell of the invention comprises human 1C3 PSMA-CAR and PD1-CTM-CD 28. In one embodiment, the modified cell of the invention comprises human 2a10 PSMA-CAR and PD1-CTM-CD 28. In one embodiment, the modified cell of the invention comprises human 2F5 PSMA-CAR and PD1-CTM-CD 28. In one embodiment, the modified cell of the invention comprises human 2C6 PSMA-CAR and PD1-CTM-CD 28. In one embodiment, the modified cells of the invention comprise murine J591 PSMA-CAR and PD1^A132LPTM-CD 28. In one embodiment, the modified cells of the invention comprise a humanized J591 PSMA-CAR and PD1^A132LPTM-CD 28. In one embodiment, the modified cells of the invention include human 1C3 PSMA-CAR and PD1 ^A132LPTM-CD 28. In one embodiment, the modified cells of the invention include human 2a10 PSMA-CAR and PD1^A132LPTM-CD 28. In one embodiment, the modified cells of the invention include human 2F5 PSMA-CAR and PD1^A132LPTM-CD 28. In one embodiment, the modified cells of the invention include human 2C6 PSMA-CAR and PD1^A132LPTM-CD 28. In one embodiment, the modified cells of the invention comprise murine J591 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cells of the invention comprise humanized J591 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cell of the invention comprises human 1C3 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cell of the invention comprises human 2a10 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cell of the invention comprises human 2F5 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cell of the invention comprises human 2C6 PSMA-CAR and TIM3-CD 28. In one embodiment, the modified cell of the invention comprises murine J591 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cells of the invention comprise humanization J591 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cell of the invention comprises human 1C3 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cell of the invention comprises human 2a10 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cell of the invention comprises human 2F5 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cell of the invention comprises human 2C6 PSMA-CAR and PD1-4-1 BB. In one embodiment, the modified cells of the invention comprise murine J591 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention comprise a humanized J591 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention include human 1C3 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention include human 2a10 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention include human 2F5 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention include human 2C6 PSMA-CAR and PD1^A132L-4-1 BB. In one embodiment, the modified cells of the invention include murine J591 PSMA-CAR and TGF β R-IL12R β 1. In one embodiment, the modified cells of the invention include a humanized J591 PSMA-CAR and TGF β 0R-IL12R β 11. In one embodiment, the modified cell of the invention comprises a human 1C3 PSMA-CAR and TGF β 2R-IL12R β 31. In one embodiment, the modified cells of the invention include human 2A10 PSMA-CAR and TGF β 4R-IL12R β 51. In one embodiment, the modified cell of the invention comprises human 2F5 PSMA-CAR and TGF β 6R-IL12R β 71. In one embodiment, the modified cells of the invention include human 2C6 PSMA-CAR and TGF β R-IL12R β 1. In one embodiment, the modified cells of the invention include murine J591 PSMA-CAR and TGF β R-IL12R β 2. In one embodiment, the modified cells of the invention include a humanized J591 PSMA-CAR and TGF β R-IL12R β 2. In one embodiment, the modified cells of the invention include human 1C3 PSMA-CAR and TGF β R-IL12R β 2. In one embodiment, the modified cells of the invention include human 2 A10 PSMA-CAR and TGF β R-IL12R β 2. In one embodiment, the modified cells of the invention include human 2F5 PSMA-CAR and TGF β R-IL12R β 2. In one embodiment, the modified cells of the invention include human 2C6 PSMA-CAR and TGF β R-IL12R β 2. Such modified cells (e.g., modified T cells), in addition to having affinity for PSMA on target cells, are capable of converting inhibitory PD-1 or TGF β signals from the microenvironment into positive (e.g., activating) signals within the modified cells. Such modified cells (e.g., modified T cells), in addition to having affinity for PSMA on target cells, are capable of converting inhibitory PD-1 or TIM-3 signaling from the microenvironment into positive (e.g., activating) CD28 signaling within the modified cells.

In exemplary embodiments, the modified cells of the invention comprise a nucleic acid encoding a bispecific antibody. In one embodiment, such modified cells can secrete the bispecific antibody outside the modified cell. In one embodiment, the modified cells of the invention comprise a nucleic acid encoding a bispecific antibody, wherein the bispecific antibody comprises more than one antigen binding domain, wherein at least one antigen binding domain binds to a negative signaling molecule (e.g., a negative signaling molecule found in the microenvironment of the modified cell), and at least one antigen binding domain binds to a costimulatory molecule on the surface of the modified cell. In one embodiment, the modified cell of the invention comprises a nucleic acid encoding a 13G4-1211 PD-L1/CD28 bispecific antibody as described herein. In one embodiment, the modified cell of the invention comprises a nucleic acid encoding a10 a5-1412 PD-L1/CD28 bispecific antibody as described herein. In one embodiment, the modified cell of the invention comprises a nucleic acid encoding a 1B12-1412 PD-L1/CD28 bispecific antibody as described herein. In one embodiment, the modified cells of the invention comprise nucleic acids encoding a TGF-beta R-1-1412 TGF-beta RII/CD28 bispecific antibody as described herein. In one embodiment, the modified cells of the invention comprise nucleic acids encoding a TGF-beta R-3-1412 TGF-beta RII/CD28 bispecific antibody as described herein.

In exemplary embodiments, the modified cells of the invention comprise a PSMA-CAR, a dominant negative receptor, and/or a switch receptor, and may further comprise a nucleic acid encoding a bispecific antibody. In addition to having affinity for PSMA on target cells, such modified cells (e.g., modified T cells) are also capable of reducing inhibitory signals from and secreting bispecific antibodies into the microenvironment in which they are present. In such cells, the activity of the bispecific antibody can further increase the activation of the modified cell (e.g., a modified T cell). In one embodiment, the modified cell of the invention comprises a PSMA-CAR selected from the group consisting of: murine J591 PSMA-CAR, humanized J591 PSMA-CAR, human 1C3 PSMA-CAR, human 2A10 PSMA-CAR, human 2F5 PSMA-CAR, and human 2C6 PSMA-CAR; TGF beta RII-DN; and expressing and secreting a bispecific antibody selected from the group consisting of: 13G4-1211 PD-L1/CD28 bispecific antibody, 10A5-1412 PD-L1/CD28 bispecific antibody, 1B12-1412 PD-L1/CD28 bispecific antibody, TGF beta R-1-1412 TGF beta RII/CD28 bispecific antibody, and TGF beta R-3-1412 TGF beta RII/CD28 bispecific antibody.

In exemplary embodiments, the modified cells of the invention comprise a PSMA-CAR, a switch receptor, and may further comprise a nucleic acid encoding a bispecific antibody. In addition to having affinity for PSMA on target cells, such modified cells (e.g., modified T cells) are also capable of converting an inhibitory signal from the microenvironment in which they are present to a positive (e.g., activating) signal within the modified cell and secreting the bispecific antibody into the microenvironment in which it is present. In such cells, the activity of the bispecific antibody can further increase the activation of the modified cell (e.g., a modified T cell). In one embodiment, the modified cell of the invention comprises a PSMA-CAR selected from the group consisting of: murine J591 PSMA-CAR, humanized J591 PSMA-CAR, human 1C3 PSMA-CAR, human 2A10 PSMA-CAR, human 2F5 PSMA-CAR, and human 2C6 PSMA-CAR; and a switch receptor selected from: PD1-CTM-CD28 switch receptor, PD1a132L-PTM-CD28 switch receptor, and TIM3-CD28 switch receptor; and expressing and secreting a bispecific antibody selected from the group consisting of: 13G4-1211 PD-L1/CD28 bispecific antibody, 10A5-1412 PD-L1/CD28 bispecific antibody, 1B12-1412 PD-L1/CD28 bispecific antibody, TGF beta R-1-1412TGF beta RII/CD28 bispecific antibody, and TGF beta R-3-1412TGF beta RII/CD28 bispecific antibody.

Any modified cell comprising a PSMA-CAR of the invention, a dominant negative receptor and/or a transducible receptor of the invention, and/or expressing and secreting a bispecific antibody of the invention is contemplated and, in view of the disclosure herein, is readily understood and made by those of skill in the art.

Methods of producing modified immune cells

The invention provides methods of producing or producing modified immune cells or precursors thereof (e.g., T cells) of the invention for use in tumor immunotherapy, such as adoptive immunotherapy. The cells are typically engineered by introducing one or more nucleic acids encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof.

In some embodiments, one or more nucleic acids encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody are introduced into the cell by an expression vector. Provided herein are expression vectors comprising one or more nucleic acid sequences of the invention encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof. Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated viral (AAV) vectors, adenoviral vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon-mediated vectors such as sleeping beauty, Piggybak, and integras (such as Phi 31). Some other suitable expression vectors include Herpes Simplex Virus (HSV) and retroviral expression vectors.

Adenoviral expression vectors are based on adenoviruses, which have low energy for integration into genomic DNA but high efficiency for transfection into host cells. The adenoviral expression vector contains sufficient adenoviral sequences to (a) support the packaging of the expression vector and (b) ultimately express the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof, in a host cell. In some embodiments, the adenoviral genome is a 36kb, linear, double-stranded DNA, wherein foreign DNA sequences (e.g., nucleic acids encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof) can be inserted in place of a large piece of adenoviral DNA to prepare the expression vectors of the invention (see, e.g., Danthinne and imperial, Gene Therapy (2000)7(20): 1707-1714).

Another expression vector is based on adeno-associated virus, which utilizes an adenovirus-coupled system. The AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thereby making it useful for gene delivery into mammalian cells, e.g., in tissue culture or in vivo. AAV vectors have a broad host range for infectivity. Details relating to the generation and use of AAV vectors are described in U.S. Pat. nos. 5,139,941 and 4,797,368.

Retroviral expression vectors are capable of integrating into the host genome, delivering large amounts of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in specialized cell lines. Retroviral vectors are constructed by inserting nucleic acids (e.g., nucleic acids encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof) into the viral genome at certain positions to produce replication-deficient viruses. Although retroviral vectors are capable of infecting a variety of cell types, integration and stable expression of the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies, and/or combinations thereof, requires division of the host cell.

Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env (see, e.g., U.S. Pat. nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been created by multiple attenuation of HIV virulence genes, for example, deletion of genes env, vif, vpr, vpu and nef, rendering the vectors biologically safe. Lentiviral vectors are capable of infecting non-dividing cells, and can be used for in vivo and ex vivo gene transfer and expression, such as of nucleic acids encoding the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies, and/or combinations thereof (see, e.g., U.S. Pat. No. 5,994,136).

Expression vectors comprising a nucleic acid of the present disclosure can be introduced into host cells by any method known to those skilled in the art. If desired, the expression vector may include viral sequences for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. Host cells can be grown and expanded in culture prior to introduction of the expression vector, followed by appropriate processing to introduce and integrate the vector. The host cells can then be expanded and can be screened for using the markers present in the vector. Various markers known in the art may be used, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or a precursor thereof, such as a T cell, NK cell, or NKT cell.

The invention also provides genetically engineered cells comprising and stably expressing the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies of the present disclosure, and/or combinations thereof. In some embodiments, the genetically engineered cell is a genetically engineered T lymphocyte (T cell), a naive T cell (TN), a memory T cell (e.g., a central memory T Cell (TCM), an effector memory cell (TEM)), a natural killer cell (NK cell), and a macrophage capable of producing progeny associated with the treatment. In one embodiment, the genetically engineered cell is an autologous cell.

The modified cells (e.g., including the subject CAR, dominant negative receptor, and/or switch receptor, and/or expressing and secreting bispecific antibody, and/or combinations thereof) can be produced by stably transfecting a host cell with an expression vector including a nucleic acid of the present disclosure. Additional methods of producing modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, light transformation, gene electrotransfer, and/or hydrodynamic delivery), and/or particle-based methods (e.g., immunoplefection, using a gene gun, and/or magnetic transfection). Transfected cells expressing the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies, and/or combinations thereof, of the present disclosure can be expanded ex vivo.

Physical methods for introducing expression vectors into host cells include calcium phosphate precipitation, lipofection, particle bombardment (particle bombardent), microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are known in the art. See, e.g., Sambrook et al (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing expression vectors into host cells include colloidally dispersed systems (e.g., macromolecular complexes, nanocapsules, microspheres, beads) and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes).

Lipids suitable for use may be obtained from commercial sources. For example, myristyl phosphatidylcholine ("DMPC") is available from Sigma, st.louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") is available from Calbiochem-Behring; myristicanyl phosphatidylglycerol esters ("DMPG") and other Lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about-20 ℃. Chloroform is used as the only solvent because it evaporates more readily than methanol. "liposomes" is a general term that encompasses a variety of mono-and multilamellar lipid carriers formed by the creation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form automatically. Prior to the formation of the closed structure, the lipid component undergoes self-rearrangement and traps water and dissolved solutes between lipid bilayers (Ghosh et al, 1991Glycobiology 5: 505-10). Also included are compositions having a structure in solution that is different from the structure of normal vesicles. For example, lipids may exhibit a micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

Whether used for introducing exogenous nucleic acid into host cells or otherwise exposing cells to the inhibitors of the invention, in order to confirm the presence of nucleic acid in host cells, a variety of assays can be performed. Such assays include, for example, molecular biological assays known to those skilled in the art (e.g., southern and northern blots, RT-PCR, and PCR); biochemical assays, such as detecting the presence or absence of a particular peptide, such as by immunological methods (ELISA and western blotting), or by assays described herein, to identify reagents that fall within the scope of the invention.

In one embodiment, the nucleic acid introduced into the host cell is RNA. In another embodiment, the RNA is mRNA, which comprises in vitro transcribed RNA or synthetic RNA. RNA can be produced by in vitro transcription using a template generated by Polymerase Chain Reaction (PCR). Target DNA from any source can be directly converted by PCR to template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable source of DNA.

PCR can be used to generate a template for in vitro transcription of mRNA, which is then introduced into the cell. Methods for performing PCR are known in the art. Primers used for PCR are designed to have regions substantially complementary to regions of DNA that can serve as templates for PCR. As used herein, "substantially complementary" refers to a nucleotide sequence that is complementary to most or all of the bases in the primer sequence. The substantially complementary sequences are capable of renaturing or hybridizing to the intended DNA target under the renaturation conditions used for PCR. The primer can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify portions of genes (open reading frames) that are normally transcribed in cells, including the 5 'and 3' UTRs. Primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, primers are designed to amplify coding regions of human cDNA, including all or part of the 5 'and 3' UTRs. Primers useful for PCR can be generated by synthetic methods known in the art. "Forward primer" refers to a primer that contains a region of nucleotides that is substantially complementary to a nucleotide on the DNA template upstream of the DNA sequence to be amplified. "upstream" is used herein to refer to the 5' position relative to the DNA sequence to be amplified in the coding strand. "reverse primer" refers to a primer that contains a nucleotide region that is substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified. "downstream" is used herein to refer to a 3' position relative to the DNA sequence to be amplified in the coding strand.

Chemical structures with the ability to promote RNA stability and/or translation efficiency may also be used. The RNA preferably has 5 'and 3' UTRs. In one embodiment, the 5' UTR is 0 to 3000 nucleotides in length. The length of the 5 'and 3' UTR sequences to be added to the coding region can be varied by different methods, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this method, one skilled in the art can modify the length of the 5 'and 3' UTRs required to achieve optimal translational efficiency following transfection of transcribed RNA.

The 5 'and 3' UTRs may be naturally occurring endogenous 5 'and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the target gene may be added by incorporating UTR sequences in the forward and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the target gene may be used to modify the stability and/or translation efficiency of the RNA. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Thus, the 3' UTR is selected or designed based on UTR properties well known in the art to increase the stability of the transcribed RNA.

In one embodiment, the 5' UTR may contain a Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the target gene is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. Kozak sequences may improve the translation efficiency of certain RNA transcripts, but it does not appear that all RNAs require such sequences for efficient translation. It is known in the art that there are many mrnas that require Kozak sequences. In other embodiments, the 5' UTR may be derived from an RNA virus, the RNA genome of which is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3 'or 5' UTRs to prevent exonuclease degradation of the mRNA.

In order to be able to synthesize RNA from a DNA template without the need for gene cloning, the transcription promoter should be ligated to the DNA template upstream of the sequence to be transcribed. When a sequence serving as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

In one embodiment, the mRNA has a cap on the 5 'end and a 3' poly (a) tail, which determines ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemer products, which are not suitable for expression in eukaryotic cells. Linearization of transcripts of plasmid DNA at the 3' UTR end results in normal size mrnas, which even if polyadenylated after transcription, are not effective in eukaryotic cell transfection.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res.,13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. biochem.,270:1485-65 (2003)).

The poly (A) tail also provides stability to the RNA and reduces its degradation, in general, the length of the poly (A) tail is positively correlated with the stability of the transcribed RNA, in one embodiment, the poly (A) tail is 100 to 5000 adenosines.

After in vitro transcription using a poly (A) polymerase, such as E.coli poly A polymerase (E-PAP), the poly (A) tail of the RNA may be further extended. In one embodiment, increasing the length of the poly (a) tail from 100 nucleotides to 300 to 400 nucleotides results in an increase in the translation efficiency of the RNA of about 2-fold. In addition, attaching different chemical groups to the 3' end increases mRNA stability. Such attachments may contain modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly (a) tail using a poly (a) polymerase. ATP analogs can further increase the stability of RNA.

The 5' cap also provides stability to the RNA molecule. In preferred embodiments, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al, Trends in biochem. Sci.,29:436-444 (2001); Stepinski, et al, RNA,7:1468-95 (2001); Elango, et al, Biochim. Biophys. Res. Commun.,330:958-966 (2005)).

In some embodiments, the RNA is electroporated into the cell, e.g., an in vitro transcribed RNA. Any solute suitable for electroporation of cells may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

In some embodiments, the nucleic acids of the present disclosure encoding the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies, and/or combinations thereof will be RNAs, e.g., RNAs synthesized in vitro. Methods for in vitro synthesis of RNA are known in the art; RNA including sequences encoding the subject CARs, dominant negative receptors and/or switch receptors, and/or bispecific antibodies, and/or combinations thereof, can be synthesized using any known method. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al cancer Res (2010)15: 9053. Introduction of an RNA comprising a nucleotide sequence encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof, into a host cell can be performed in vitro, ex vivo, or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with an RNA that includes a nucleotide sequence encoding the subject CAR, a dominant negative receptor and/or a switch receptor, and/or a bispecific antibody, and/or a combination thereof.

The methods of the present disclosure can be applied to modulate T cell activity in basic research and therapy in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill target cancer cells.

The method also provides the ability to control expression levels over a wide range by altering, for example, the promoter or amount of input RNA, such that it is capable of independently regulating expression levels. In addition, PCR-based mRNA generation techniques greatly facilitate the design of mrnas with different structures and combinations of their domains.

One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not require a vector. The RNA transgene can be delivered to lymphocytes and expressed therein after brief in vitro cell activation as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is not possible. Due to the transfection efficiency of RNA and its ability to uniformly modify the entire lymphocyte population, cloning of the cells is not necessary.

Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been successfully tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications to achieve prolonged expression of transferred IVT-RNA.

Certain IVT vectors are known in the literature, which are used in a standardized manner as templates for in vitro transcription and are genetically modified in such a way that stable RNA transcripts are produced. Currently, the protocols used in the art are based on plasmid vectors having the following structure: a 5'RNA polymerase promoter capable of transcribing RNA, followed by a gene of interest flanked 3' and/or 5 'by an untranslated region (UTR), and a 3' poly-adenine box containing 50 to 70 a nucleotides. Before in vitro transcription, the circular plasmid is linearized downstream of the poly-adenine-based cassette by a type II restriction enzyme (recognition sequence corresponds to the cleavage site). Thus, the poly-adenine-based box corresponds to the latter poly (A) sequence in the transcript. Due to this procedure, some nucleotides remain part of the cleavage site after linearization and extend or mask the poly (A) sequence at the 3' end. It is not clear whether such non-physiological projections affect the amount of protein produced intracellularly by such constructs.

In another aspect, the RNA construct is delivered into the cell by electroporation. See, e.g., formulations and methods for electroporation of nucleic acid constructs into mammalian cells as taught in US 2004/0014645, US 2005/0052630a1, US 2005/0070841a1, US 2004/0059285a1, US 2004/0092907a 1. Various parameters including the electric field strength required for electroporation of any known cell type are generally known in the relevant research literature and numerous patents and applications in the field. See, e.g., U.S. patent No. 6,678,556, U.S. patent No. 7,171,264, and U.S. patent No. 7,173,116. Commercially available devices for therapeutic application of electroporation, e.g., MedPulser ^TMDNA polymerization Therapy systems (inovoio/genetics, San Diego, Calif.) and they have been described in numerous patents, such as U.S. patent nos. 6,567,694; U.S. patent No. 6,516,223, U.S. patent No. 5,993,434, U.S. patent No. 6,181,964, U.S. patent No. 6,241,701, and U.S. patent No. 6,233,482; electroporation can also be used to transfect cells in vitro, as described in US20070128708a 1. Electroporation can also be used to deliver nucleic acids into cells in vitro. Thus, electroporation-mediated administration of nucleic acids (including expression constructs) into cells using any of the many available devices and electroporation systems known to those skilled in the art provides an exciting new means of delivering target RNA to target cells.

In some embodiments, immune cells (e.g., T cells) can be incubated or cultured prior to, during, and/or subsequent to introduction of a nucleic acid molecule encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof. In some embodiments, the cells (e.g., T cells) can be incubated or cultured prior to, during, and/or subsequent to introduction of the nucleic acid molecule encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof, such as prior to, during, or subsequent to transduction of the cells with a viral vector (e.g., lentiviral vector) encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof. In some embodiments, the methods comprise activating or stimulating the cell with a stimulating or activating agent (e.g., an anti-CD 3/anti-CD 28 antibody) prior to introducing the nucleic acid molecule encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof.

In some embodiments, when the nucleic acid sequences of the invention encoding the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof are present on one or more separate nucleic acid sequences, the order of introduction of each of the one or more nucleic acid sequences can be altered. For example, a nucleic acid sequence encoding the subject CAR and dominant negative receptor and/or switch receptor can be introduced into a host cell first, followed by introduction of a nucleic acid sequence encoding a bispecific antibody. For example, a nucleic acid sequence encoding a bispecific antibody can be first introduced into a host cell, followed by introduction of a nucleic acid sequence encoding the subject CAR and a dominant negative receptor and/or switch receptor. In some embodiments, each of the one or more nucleic acid sequences is introduced into the host cell simultaneously. One skilled in the art can determine the order in which each of the one or more nucleic acid sequences is introduced into the host cell.

H. Sources of immune cells

Prior to expansion, a source of immune cells is obtained from the subject for ex vivo manipulation. Sources of target cells for ex vivo procedures may also include, for example, autologous or allogeneic donor blood, cord blood, or bone marrow. For example, the source of immune cells can be from a subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's umbilical cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells that are innate or adaptive immune, e.g., myeloid (myeloid) cells or lymphoid (lymphoid) cells (including lymphocytes, typically T cells and/or NK cells). Other exemplary cells include stem cells, such as pluripotent (multipotent) stem cells and pluripotent (pluripotent) stem cells, including induced pluripotent stem cells (ipscs). In some aspects, the cell is a human cell. With respect to the subject to be treated, the cells may be allogeneic and/or autologous. The cells are typically primary cells, such as cells isolated directly from a subject and/or isolated from a subject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and/or a basophil. In embodiments, the target cell is an Induced Pluripotent Stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell produced by a subject, manipulated to alter (e.g., induce mutations) or manipulate expression of one or more target genes and differentiate into e.g., a T cell, such as a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

In some embodiments, the cells comprise one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and subsets thereof, such as those defined by function, activation state, maturity, differentiation capacity, expansion, circulation, localization, and/or persistence capacity, antigen specificity, antigen receptor type (present in a particular organ or compartment), marker or cytokine secretion profile, and/or degree of differentiation. Among these subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells are naive T (tn) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (tscm), central memory T (tcm), effector memory T (tem) or terminally differentiated effector memory T cells, Tumor Infiltrating Lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (mait) cells, naturally occurring and adaptive regulatory T (treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and/gamma T cells). In certain embodiments, any number of T cell lines available in the art may be used.

In some embodiments, the methods comprise isolating immune cells from a subject, preparing, processing, culturing, and/or engineering them. In some embodiments, the preparation of the engineered cell comprises one or more culturing and/or preparation steps. The cells for the described engineering can be isolated from a sample, such as a biological sample, e.g., a sample obtained from, or derived from, a subject. In some embodiments, the subject from which the cells are isolated is a subject having a disease or disorder, or in need of cell therapy, or to which cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, processed, and/or engineered. Thus, in some embodiments, the cell is a primary cell, such as a primary human cell. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples from one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a processed sample. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is derived from the product of apheresis or apheresis (leukapheresis). Exemplary samples include whole blood, Peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, small intestine, large intestine, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs, and/or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy), samples include samples from both autologous and allogeneic sources.

In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, e.g., from mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells comprises one or more preparative and/or cell-based non-affinity isolation steps. In some examples, for example, cells are washed, centrifuged, and/or cultured in the presence of one or more reagents to remove unwanted components, enrich for desired components, lyse, or remove cells that are sensitive to a particular reagent. In some examples, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity, and/or resistance to a particular component.

In some examples, cells from the circulating blood of the subject are obtained, such as by apheresis or apheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects, the sample contains other cells in addition to erythrocytes and platelets. In some embodiments, the blood cells collected by the subject are washed to remove the plasma fraction and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some aspects, the washing step is accomplished by Tangential Flow Filtration (TFF) according to manufacturer's instructions. In some embodiments, after washing, the cells are resuspended in a variety of biocompatible buffers. In certain embodiments, the blood cell sample is removed from the culture medium and resuspended directly in the culture medium. In some embodiments, the methods include density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing erythrocytes and centrifuging through a Percoll or Ficoll gradient.

In one embodiment, the immune cells from the circulating blood of the individual are obtained by apheresis or apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and/or platelets. Cells collected by apheresis may be washed to remove the plasma fraction and the cells placed in a suitable buffer or culture medium (such as Phosphate Buffered Saline (PBS) or, the wash solution may be calcium-free or may be magnesium-free or may be free of many but not all divalent cations) for subsequent processing steps. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesired components of apheresis may be removed and the cells resuspended directly in culture medium.

In some embodiments, the isolation methods comprise isolating different cell types based on expression or the presence of one or more specific molecules in the cell, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such labeling may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, in some aspects, isolation includes isolation of cells and cell populations based on the expression level of cells or one or more markers (typically cell surface markers), e.g., by incubation with an antibody or binding partner that specifically binds such markers, followed by typically a washing step and separation of cells that have bound the antibody or binding partner from those that have not bound the antibody or binding partner.

Such isolation steps may be based on positive selection, in which cells bound to the agent are retained for further use, and/or negative selection, in which cells not bound to the antibody or binding partner are retained. In some instances, both portions are reserved for further use. In some aspects, negative selection is particularly useful when antibodies that specifically recognize cell types in a heterogeneous population are not available, allowing optimal isolation based on markers expressed by cells other than the desired population. Isolation need not result in 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of cells of a particular type (such as those expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal, or elimination of cells of a particular type (such as those expressing a marker) refers to a reduction in the number or percentage of such cells, but need not result in the complete removal of all such cells.

In some examples, multiple rounds of separation steps are performed, wherein the portions positively or negatively selected in one step are subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single isolation step can simultaneously eliminate cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each specific for a marker targeting a negative selection. Likewise, multiple cell types can be positively selected simultaneously by incubating the cells with antibodies or binding partners expressed on the various cell types.

In some embodiments, one or more of the enriched or depleted T cell populations is positive (marker +) for one or more particular markers (e.g., surface markers) or expresses high levels thereof (marker)^{Height of}) Or negative for one or more specific markers (marker-) Or express its relatively low level (marker)^{Is low in}) The cell of (1). For example, in some aspects, a specific subpopulation of T cells, such as T cells that are positive or express high levels of one or more surface markers (e.g., CD28+, CD62L +, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA +, and/or CD45RO +), are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and/or CD62L in enriched (i.e., positively selected) cells (such as CD8+ cells, or T cells, e.g., CD3+ cells) and/or depleted (i.e., negatively selected) cells in which are positive for or express high surface levels of CD45 RA. In some embodiments, the cells are enriched for or depleted of cells that are positive for or express high surface levels of CD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45 RA) and for CD 62L. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g.,

M-450 CD3/CD 28T cell expander).

In some embodiments, T cells are separated from the PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such populations of CD4+ and CD8+ may be further sorted into subpopulations by positive or negative selection for expression or a relatively high degree of expression of a marker on one or more populations of naive memory and/or effector T cells. In some embodiments, the CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment of central memory t (tcm) cells is performed to increase efficacy, such as to provide long-term survival, expansion, and/or transplantation after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD8+ CD62L + and CD 62L-subsets of peripheral blood lymphocytes. The PBMCs may be enriched for or depleted of CD62L-CD8+ and/or CD62L + CD8+ moieties, such as with anti-CD 8 and anti-CD 62L antibodies. In some embodiments, the population of CD4+ T cells and the CD8+ T cell subpopulation (e.g., enrichment of subpopulations of central memory (TCM) cells in some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. in some aspects, isolation of a CD8+ population of TCM-enriched cells is performed by clearing cells expressing CD4, CD14, CD45RA and positively selecting or enriching for cells expressing CD 62L. in one aspect, enrichment of central memory T (TCM) cells is performed starting with a negative portion of cells selected based on CD4 expression, which undergo negative selection based on CD14 and CD45RA expression, and positive selection based on CD 62L. in some aspects, such selections are performed simultaneously, and in any other way in sequence. In some aspects, the same CD4 expression-based selection step used in making a CD8+ cell population or subpopulation may also be used to generate a CD4+ cell population or subpopulation, such that both the positive and negative portions from CD 4-based isolation are retained and used for subsequent steps in the method, optionally after one or more positive or negative selection steps.

CD4+ T helper cells were sorted into naive, central memory and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, the naive CD4+ T lymphocyte is a CD45RO-, CD45RA +, CD62L +, CD4+ T cell. In some embodiments, the central memory CD4+ cells are CD62L + and CD45RO +. In some embodiments, the effector CD4+ cells are CD62L "and CD45 RO. In one example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD 8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic or paramagnetic bead, allowing for the separation of positive and/or negative selection of cells.

In some embodiments, the cells are incubated and/or cultured prior to or in conjunction with genetic engineering. The incubation step comprises culturing, incubating, stimulating, activating and/or propagating. In some embodiments, the composition or cell is incubated in the presence of a stimulatory condition or a stimulatory agent. Such conditions include those designed to induce cell proliferation, expansion, activation, and/or survival in a population, mimic antigen exposure, and/or prime cells for genetic engineering, such as to introduce recombinant antigen receptors. Conditions may include one or more of a particular culture medium, temperature, oxygen content, carbon dioxide content, time, reagents (e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other reagent designed to activate cells). In some embodiments, the stimulating condition or agent comprises one or more agents, such as a ligand capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent opens or initiates a TCR/CD3 intracellular signaling cascade in a T cell. Such agents may include antibodies, such as those specific for TCR components and/or co-stimulatory receptors (e.g., anti-CD 3, anti-CD 28), e.g., bound to a solid support, such as a bead, and/or one or more cytokines. Optionally, the amplification method may further comprise the step of adding anti-CD 3 and/or anti-CD 28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agent includes IL-2 and/or IL-15, e.g., an IL-2 concentration of at least about 10 units/mL.

In another embodiment, the red blood cells are lysed and mononuclear cells are removed from the red blood cellsIsolation of T cells from peripheral blood, e.g. by PERCOLL^TMThe gradient was centrifuged. Alternatively, the T cells can be isolated from the umbilical cord. In any case, specific subpopulations of T cells may be further isolated by positive or negative selection techniques.

Cells expressing certain antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56, can be eliminated from the cord blood mononuclear cells so isolated. The removal of these cells can be accomplished using isolated antibodies, biological samples comprising antibodies (such as ascites fluid), antibodies bound to a physical support, and cells bound to antibodies.

Enrichment of the T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. Preferred methods are cell sorting and/or cell selection via negative magnetic immunoadhesion or flow cytometry using mixtures of monoclonal antibodies directed to cell surface markers presented on negatively selected cells. For example, to enrich for CD4 by negative selection⁺Cells, monoclonal antibody mixtures typically include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD 8.

For isolation of a desired cell population by positive or negative selection, the concentration and surface (e.g., particles such as beads) of the cells can be varied. In certain embodiments, it is desirable to significantly reduce the volume of mixing the beads and cells together (i.e., increase the concentration of cells), thereby ensuring maximum contact of the cells and beads. For example, in one embodiment, a concentration of 20 hundred million cells/ml is used. In one embodiment, a concentration of 10 hundred million cells/ml is used. In a further embodiment, greater than 1 hundred million cells/ml are used. In further embodiments, the concentration of cells used is ten million, fifteen million, twenty five million, thirty five million, forty five million, or fifty million cells/ml. In yet another embodiment, the concentration of cells used is seventy-five million, eighty-five million, ninety-ten million, ninety-five million, or one hundred million cells/ml. In further embodiments, concentrations of one hundred and twenty million or one hundred and fifty million cells per ml may be used. Increased cell yield, cell activation, and cell expansion can be achieved using high concentrations.

After the washing step, the T cells can also be frozen, which does not require a monocyte removal step. While not wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and a degree of monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing fluid. While a number of freezing fluids and parameters are known in the art and may be useful in the present context, in a non-limiting example, one approach involves the use of PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells were then frozen at a rate of 1 ℃ per minute to-80 ℃ and stored in the gas phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as immediate uncontrolled freezing at-20 ℃ or in liquid nitrogen.

In one embodiment, the T cell population is included within cells, such as peripheral blood mononuclear cells, cord blood cells, purified T cell populations, and T cell lines. In another embodiment, the peripheral blood mononuclear cells comprise a population of T cells. In yet another embodiment, the purified T cells comprise a population of T cells.

In certain embodiments, T regulatory cells (Tregs) can be isolated from a sample. The sample may include, but is not limited to, cord blood or peripheral blood. In certain embodiments, the Tregs are isolated by flow cytometry sorting. The Tregs in the sample can be enriched and then isolated by any means known in the art. Prior to use, the isolated Tregs may be cryopreserved and/or expanded. Methods of isolating Tregs are described in U.S. patent nos. 7,754,482, 8,722,400, and 9,555,105 and U.S. patent application No. 13/639,927, the contents of which are incorporated herein in their entirety.

I. Expansion of immune cells

Whether before or after the cells are modified to express the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or combinations thereof, the cells can be activated and expanded using methods described in the following references, e.g., U.S. patent nos. 6,352,694; 6,534,055, respectively; 6,905,680, respectively; 6,692,964, respectively; 5,858,358, respectively; 6,887,466, respectively; 6,905,681, respectively; 7,144,575, respectively; 7,067,318, respectively; 7,172,869, respectively; 7,232,566, respectively; 7,175,843, respectively; 5,883,223, respectively; 6,905,874, respectively; 6,797,514, respectively; 6,867,041, respectively; and U.S. patent application No. 20060121005. For example, the T cells of the invention may be expanded by contact with a surface to which have been attached an agent that stimulates a signal associated with the CD3/TCR complex and a ligand that stimulates a co-stimulatory molecule on the surface of the T cell. Specifically, the T cell population may be stimulated by contact with: an anti-CD 3 antibody or antigen-binding fragment thereof, or an anti-CD 2 antibody immobilized on a surface, or an activator of protein kinase C (e.g., bryodin) bound to a calcium ionophore. To co-stimulate accessory molecules on the surface of T cells, ligands that bind the accessory molecules are used. For example, T cells may be contacted with an anti-CD 3 antibody and an anti-CD 28 antibody under conditions suitable to stimulate T cell proliferation. Examples of anti-CD 28 antibodies include 9.3, B-T3, XR-CD28(Diaclone, Besancon, France) and these can be used in the present invention as can other methods and reagents known in the art (see, e.g., ten Berge et al, Transplant Proc. (1998)30(8): 3975; Haanen et al, J.Exp.Med. (1999)190(9): 1319-.

T cells amplified by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, as well as any and all whole or partial integers therebetween. In one embodiment, the T cell expansion ranges from about 20 fold to about 50 fold.

After culturing, the T cells may be incubated in cell culture medium in a culture device for a period of time or until the cells reach a high cell concentration for confluency or optimal passage, and then transferred to another culture device. The culture device may be any culture device commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater prior to transferring the cells to another culture device. More preferably, the confluence level is 90% or greater. The period of time may be any time suitable for culturing cells in vitro. The T cell medium may be replaced at any time during the culturing of the T cells. Preferably, the T cell culture medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture device and can then be used immediately or cryopreserved for use at a later time. In one embodiment, the invention includes cryopreserving expanded T cells. Cryopreserved T cells are thawed prior to introducing nucleic acid into the T cells.

In another embodiment, the method comprises isolating T cells and expanding T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, cryopreserved T cells are thawed for electroporation using RNA encoding the chimeric membrane protein.

Another process for ex vivo expansion of cells is described in U.S. patent No. 5,199,942, incorporated herein by reference. Amplification such as that described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other amplification methods described herein. Briefly, ex vivo culture and expansion of T cells involves the addition of cell growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

The culturing step described herein (either in contact with the reagents described herein or after electroporation) can be very short, e.g., less than 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step(s) (contacting with the reagents described herein) further described herein can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell cultures generally refer to cells taken from living organisms and grown under controlled conditions. A primary cell culture is a culture taken directly from a cell, tissue or organ of an organism and prior to a first passage culture. When cells are placed in a growth medium under conditions that promote cell growth and/or division, the cells in the culture are expanded, resulting in a larger cell population. When expanding cells in culture, the rate of cell proliferation is typically measured by the amount of time required for the number of cells to double, also known as doubling time.

Each round of subculture is called a passage. When cells are subcultured, they are said to have been passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e. the first culture after isolation of cells from the tissue, was designated P0. After the first passage culture, the cells were described as a secondary culture (P1 or passage 1). After the second culture, the cells became the third culture (P2 or passage 2), and so on. Those skilled in the art will appreciate that there are many population doublings in the passage cycle; thus, the number of culture population doublings is greater than the number of passages. The expansion of cells (i.e., the number of population doublings) in the period between passages depends on many factors, including but not limited to, seeding density, substrate, culture medium, and time between passages.

In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hour-wise integer in between. Suitable conditions for T cell culture include suitable Media (e.g., mineral Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN- γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF- β, and TNF- α, or any other additive known to those skilled in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma preparations (plasmates), and reductantsAgents such as N-acetyl-cysteine and 2 mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo 15, and X-Vivo 20, an Optimizer, as well as added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined group of hormones, and/or cytokine(s) in amounts sufficient for T-cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in the experimental culture and are not included in the cell culture to be injected into the subject. The target cells are maintained under conditions necessary to support growth, e.g., at an appropriate temperature (e.g., 37 ℃) and atmospheric air (e.g., air plus 5% CO) ₂)。

The medium used to culture the T cells may include agents that can co-stimulate the T cells. For example, the agent that can stimulate CD3 is an antibody to CD3, and the agent that can stimulate CD28 is an antibody to CD 28. Cells isolated by the methods described herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000, 10,000,000-fold, or greater. In one embodiment, the T cell expansion ranges from about 20 fold to about 50 fold, or more. In one embodiment, human T regulatory cells are expanded via KT64.86 artificial antigen presenting cells (aapcs) coated with anti-CD 3 antibody. In one embodiment, human T regulatory cells are expanded via K562 artificial antigen presenting cells (aapcs) coated with anti-CD 3 antibody. Methods of expanding and activating T cells can be found in U.S. patent nos. 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

In one embodiment, the method of expanding T cells may further comprise isolating the expanded T cells for further use. In another embodiment, the method of expanding may further comprise subsequently electroporating the expanded T cells prior to culturing. Subsequent electroporation can include introducing nucleic acid encoding the agent (such as transduced amplified T cells with the nucleic acid, transfected amplified T cells, or electroporated amplified T cells) into the expanded T cell population, wherein the agent further stimulates the T cells. The agent may stimulate the T cell, such as by stimulating further expansion, effector function, or another T cell function.

J. Method of treatment

Modified cells (e.g., T cells) described herein can be included in compositions for immunotherapy. The composition may comprise a pharmaceutical composition and further comprise a pharmaceutically acceptable carrier. A therapeutically effective amount of a pharmaceutical composition comprising modified T cells may be administered.

In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified T cell of the invention. In another aspect, the invention includes a method of treating a disease or disorder in a subject comprising administering to a subject in need thereof a modified population of T cells.

Also included are methods of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a modified cell (e.g., a modified T cell) of the invention. In one embodiment, a method of treating a disease or disorder in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising the subject CAR, dominant negative receptor and/or switch receptor, and/or bispecific antibody, and/or a combination thereof. In one embodiment, a method of treating a disease or disorder in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising a subject CAR (e.g., a CAR having affinity for PSMA on a target cell) and a dominant negative receptor and/or a switch receptor. In one embodiment, a method of treating a disease or disorder in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising a subject CAR (e.g., a CAR having affinity for PSMA on a target cell) and a dominant negative receptor and/or switch receptor, and wherein the modified cell is capable of expressing and secreting a bispecific antibody.

Methods of administering immune cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, adoptive T cell therapy is described as follows: U.S. patent application publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. nos. 4,690,915, owned by Rosenberg; rosenberg (2011) Nat Rev Clin Oncol.8(10): 577-85). See, e.g., Themeli et al (2013) Nat Biotechnol.31(10): 928-933; tsukahara et al (2013) Biochem Biophys Res Commun 438(1) 84-9; davila et al, (2013) PLoS ONE 8(4) e 61338. In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer, where cells are isolated and/or otherwise prepared from the subject to receive the cell therapy, or from a sample derived from the subject. Thus, in some aspects, the cells are derived from a subject in need of treatment, such as a patient, and the cells are administered to the same subject after isolation and processing.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and/or otherwise prepared from a subject other than the subject (e.g., the first subject) to receive or ultimately receive cell therapy. In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype (supertype) as the first subject.

In some embodiments, the subject has been treated with a therapeutic agent that targets a disease or disorder (e.g., a tumor) prior to administration of the cells or cell-containing composition. In some aspects, the additional therapeutic agent is refractory or non-responsive to the subject. In some embodiments, the subject has a persistent or recurrent disease, e.g., after treatment with another therapeutic intervention, including chemotherapy, radiation, and/or Hematopoietic Stem Cell Transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having developed resistance to another therapy.

In some embodiments, the subject is responsive to other therapeutic agents, and treatment with the therapeutic agent reduces the burden of the disease. In some aspects, the subject is initially responsive to the therapeutic agent, but over time the subject exhibits a recurrence of the disease or disorder. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of, such as at high risk of, relapse, and the cells are therefore administered prophylactically, e.g., to reduce the likelihood of, or prevent, relapse. In some aspects, the subject has not received prior treatment (prior treatment) with another therapeutic agent.

In some embodiments, the subject has a persistent or recurrent disease, e.g., after treatment with another therapeutic intervention, including chemotherapy, radiation, and/or Hematopoietic Stem Cell Transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having developed resistance to another therapy.

The modified immune cells of the invention can be administered to an animal, preferably a mammal, more preferably a human, to treat cancer. Furthermore, the cells of the invention may be used to treat any condition associated with cancer, in particular a cell-mediated immune response against tumor cells, where treatment or alleviation of the disease is desired. The types of cancer treated with the modified cells or pharmaceutical compositions of the invention include carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancer may be a non-solid tumor (such as a hematological tumor) or a solid tumor. Also included are adult tumors/cancers and pediatric tumors/cancers.

A solid tumor is an abnormal tissue mass that generally does not include cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), Astrocytoma, CNS lymphoma, blastoblastoma, medulloblastoma, schwannoma, craniopharyngioma (craniophagiogioma), ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastasis).

Cancers for which therapy may be applicable by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular cancer, basal cell carcinoma (a kind of skin cancer), squamous cell carcinoma (various tissues), bladder cancer (including transitional cell carcinoma (bladder malignancy)), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell and non-small cell cancers of the lung), adrenocortical cancer, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary cancer, renal cell carcinoma, ductal carcinoma in situ (ductal carcinoma in situ) or bile duct cancer, choriocarcinoma, seminoma, embryonic carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma (osteogenic carcinosoma), epithelial cancer, and nasopharyngeal cancer.

Sarcomas that can be suitably treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliocytoma, synovioma, mesothelioma, ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Prostate adenocarcinoma is an extremely common and fatal disease. Prostate cancer is the most common malignancy in men. Prostate cancer is the second leading cause of cancer-related death in men, estimated to account for 10% of cancer deaths annually in men. PSMA is highly expressed in malignant prostate tissue and is low expressed at some normal human tissues. Under normal physiological conditions, PSMA is expressed in the prostate gland (secretory acinar epithelium), kidney (proximal tubule), nervous system glia (astrocytes and schwann cells), and small intestine (jejunal brush border). PSMA is more highly expressed in prostate epithelium and is significantly upregulated in malignant prostate tissue. PSMA expression in normal cells has been found to be 100-fold to 1000-fold lower than in prostate cancer cells. PSMA expression was significantly increased during the transition from benign prostatic hyperplasia to prostate adenocarcinoma. PSMA expression has been found to be directly related to the histological grade of malignant prostate tissue and increases with more advanced disease (i.e., highest PSMA expression found in prostate cancer metastasis to lymph nodes and bone).

In one embodiment, the methods of the invention are useful for treating prostate cancer, e.g., advanced castration resistant prostate cancer. One skilled in the art will readily appreciate that any type of cancer in which PSMA tumor antigen is expressed may be treated using the methods of the present invention. For example, neovascular expression of PSMA is found in non-small cell lung cancer, see, e.g., PLoS one.2017oct 27; 12(10). Thus, the methods of the invention may also be used to treat non-small cell lung cancer (NSCLC).

In certain exemplary embodiments, the modified immune cells of the invention are used to treat prostate cancer. In one embodiment, the methods of the invention are used to treat castration-resistant prostate cancer. In one embodiment, the methods of the invention are used to treat late-stage castration-resistant prostate cancer. In one embodiment, the methods of the invention are used to treat metastatic castration-resistant prostate cancer. In one embodiment, the methods of the invention are used to treat metastatic castration-resistant prostate cancer, wherein patients with metastatic castration-resistant prostate cancer have ≧ 10% PSMA-expressing tumor cells. In one embodiment, the methods of the invention are used to treat castration-resistant prostate cancer, wherein the patient has castration testosterone levels (e.g., <50ng/mL) with or without androgen deprivation therapy.

In certain embodiments, the subject is provided with secondary therapy. Secondary therapies include, but are not limited to, chemotherapy, radiation, surgery, and drugs.

The cells of the invention may be administered at dosages, routes and times determined by appropriate preclinical and clinical trials and trials. The cell composition may be administered multiple times at doses within these ranges. Administration of the cells of the invention may be combined with other methods of treating the desired disease or disorder, as determined by one of skill in the art.

For a subject receiving treatment, the cells of the invention to be administered may be autologous.

Administration of the cells of the invention may be carried out in any convenient manner known to those skilled in the art. The cells of the invention may be administered to a subject by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. The compositions described herein may be administered to a patient arterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other cases, the cells of the invention are injected directly into an inflammatory site of a subject, a localized disease site of a subject, a lymph node, an organ, a tumor, etc.

In some embodiments, the cells are administered at a desired dose, which in some aspects includes a desired dose or number of cells or cell type and/or desired ratio of cell types. Thus, in some embodiments, the dosage of cells is based on the total number of cells (or number per kg body weight) and a desired ratio of populations or subtypes of individuals, such as the ratio of CD4+ to CD8 +. In some embodiments, the dosage of cells is based on the total number of cells (or number per kg body weight) in an individual population or individual cell type. In some embodiments, the dose is based on a combination of such characteristics as the desired total number of cells, the desired ratio, and the desired total number of cells in the individual population.

In some embodiments, a population or subtype of cells (such as CD 8)⁺And CD4⁺T cells) are administered at or within a tolerance of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to which the cells are administered, such as cells/kg. In some aspects, the desired dose is at or above the minimum number of cells or the minimum number of cells per unit body weight. In some aspects, a population or subset of individuals is administered at a desired output ratio (such as CD 4) among total cells administered at a desired dose⁺And CD8⁺Ratio) or close to it, such as within some tolerance or error of the ratio.

In some embodiments, the cells are administered within tolerance or high tolerance of a desired dose of one or more of an individual population or subtype of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells or a desired number of such cells per unit body weight of the subject to which the cells are administered, such as cells/kg. In some aspects, the desired dose is at or above the minimum number of cells of the population or subtype or the minimum number of cells of the population or subtype per unit body weight. Thus, in some embodiments, the dose is based on a desired fixed dose and a desired ratio of total cells, and/or on a desired fixed dose of one or more (e.g., each) of the individual subtypes or subpopulations. Thus, in some embodiments, the dose is based on a desired fixed or minimum dose of T cells and CD4 ⁺And CD8⁺Desired ratio of cells, and/or based on CD4⁺And/or CD8⁺A desired fixed or minimum dose of cells.

In certain embodiments, the cells, or individual populations of subtypes of cells, are administered to the subject in a range of about one million to about one billion cells, such as, e.g., 100 to about 500 billion cells (e.g., about 500 trillion cells, about 2500 trillion cells, about 5 billion cells, about 10 billion cells, about 50 billion cells, about 200 billion cells, about 300 billion cells, about 400 billion cells, or a range defined by any two of the foregoing values), such as, e.g., about 1000 to about 1000 trillion cells (e.g., about 2000 trillion cells, about 3000 trillion cells, about 4000 trillion cells, about 6000 trillion cells, about 7000 trillion cells, about 8000 trillion cells, about 9000 trillion cells, about 100 trillion cells, about 250 trillion cells, about 500 trillion cells, about 750 trillion cells, about 900 trillion cells, or a range defined by any two of the above values), and in some cases from about 1 million cells to about 500 million cells (e.g., about 1.2 million cells, about 2.5 million cells, about 3.5 million cells, about 4.5 million cells, about 6.5 million cells, about 8 million cells, about 9 million cells, about 30 million cells, about 300 million cells, about 450 million cells), or any value in between these ranges.

In some embodiments, the dose of total cells and/or the dose of individual subpopulations of cells is within the following ranges: equal to or about 1x10⁵Individual cell/kg to about 1x10¹¹Individual cell/kg, 10⁴Equal to or about 10¹¹Individual cells per kilogram (kg) body weight, such as 10⁵To 10⁶Individual cells/kg body weight, e.g., equal to or about 1x10⁵Individual cell/kg, 1.5X 10⁵Individual cell/kg, 2X 10⁵Individual cell/kg, or 1x10⁶Cells/kg body weight. For example, in some embodiments, the cells are administered with a certain margin of error or range within: equal to or about 10⁴And equal to or about 10⁹Individual T cells per kilogram (kg) body weight, such as 10⁵To 10⁶Between T cells/kg body weight, e.g., equal to or about 1x10⁵1.5X 10T cells/kg⁵T cells/kg, 2X 10⁵T cells/kg, or 1x10⁶Individual T cells/kg body weight. In other exemplary embodiments, modified for use in the methods of the present disclosureSuitable doses of cells include, but are not limited to, about 1x10⁵Individual cell/kg to about 1x10⁶Individual cell/kg, about 1X10⁶Individual cell/kg to about 1x10⁷Individual cell/kg, about 1X10⁷Individual cell/kg to about 1x10⁸Individual cell/kg, about 1X10⁸Individual cell/kg to about 1x10⁹Individual cell/kg, about 1X10⁹Individual cell/kg to about 1x10 ¹⁰Individual cell/kg, about 1X10¹⁰Individual cell/kg to about 1x10¹¹Individual cells/kg. In exemplary embodiments, a suitable dose for use in the methods of the present disclosure is about 1x10⁸Individual cells/kg. In exemplary embodiments, a suitable dose for use in the methods of the present disclosure is about 1x10⁷Individual cells/kg. In other embodiments, a suitable dose is about 1x10⁷Total cells to about 5x10⁷And (4) total cells. In some embodiments, a suitable dose is about 1x10⁸Total cells to about 5x10⁸And (4) total cells. In some embodiments, a suitable dose is about 1.4x10⁷Total cells to about 1.1x10⁹And (4) total cells. In an exemplary embodiment, a suitable dose for use in the methods of the present disclosure is about 7x10⁹And (4) total cells. In an exemplary embodiment, a suitable dose is about 1x10⁷Total cells to about 3X10⁷And (4) total cells.

In some embodiments, the dose of total cells and/or the dose of individual subpopulations of cells is in the following ranges: equal to or about 1x10⁵Cell/m²To about 1x10¹¹Cell/m². In exemplary embodiments, the dose of total cells and/or the dose of individual subpopulations of cells is in the following ranges: equal to or about 1x10⁷/m²To about or about 3x10 ⁷/m². In exemplary embodiments, the dose of total cells and/or the dose of an individual subpopulation of cells is at or about 1x10⁸/m²To equal or about 3x10⁸/m²Within the range of (a). In some embodiments, the dose of total cells and/or the dose of an individual subpopulation of cells is the maximum allowable dose for a given patient.

In some embodiments, the cells are administered with a certain error range or range within: equal to or about 10⁴And equal to or about 10⁹An individual CD4⁺And/or CD8⁺Cells per kilogram (kg) body weight, such as 10⁵And 10⁶An individual CD4⁺And/or CD8⁺Between cells/kg body weight, e.g., equal to or about 1x10⁵An individual CD4⁺And/or CD8⁺Cell/kg, 1.5X 10⁵An individual CD4⁺And/or CD8⁺Cell/kg, 2X 10⁵An individual CD4⁺And/or CD8⁺Cells/kg, or 1x10⁶An individual CD4⁺And/or CD8⁺Cells/kg body weight. In some embodiments, the cells are administered with a margin or range of error greater than, and/or at least about 1x10⁶About 2.5 x10⁶About 5 x10⁶About 7.5 x10⁶Or about 9 x10⁶An individual CD4⁺Cells, and/or at least about 1x10⁶About 2.5 x10⁶About 5 x10⁶About 7.5 x10⁶Or about 9 x10⁶CD8+ cells and/or at least about 1x10 ⁶About 2.5 x 10⁶About 5 x 10⁶About 7.5 x 10⁶Or about 9 x 10⁶And (4) T cells. In some embodiments, the cells are administered with a margin or range of error greater than: about 10⁸And 10¹²Between or about 10¹⁰And 10¹¹Between T cells, about 10⁸And 10¹²Between or about 10¹⁰And 10¹¹An individual CD4⁺Between cells, and/or about 10⁸And 10¹²Between or about 10¹⁰And 10¹¹An individual CD8⁺Between individual cells.

In some embodiments, the cells are administered at or within a tolerance range of a desired output ratio for a plurality of cell populations or subtypes (such as CD4+ and CD8+ cells or subtypes). In some aspects, the desired ratio may be a specific ratio or may be a range of ratios, for example, in some embodiments, the desired ratio (e.g., CD 4)⁺And CD8⁺Ratio of cells) ofEqual to or between about 5:1 and equal to or about 5:1 (or about 1:5 and less than about 5:1), or equal to or between about 1:3 and equal to or about 3:1 (or greater than about 1:3 and less than about 3:1), such as equal to or between about 2:1 and equal to or about 1:5 (or greater than about 1:5 and less than about 2:1), such as equal to or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1: 5. In some aspects, the tolerance is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

In some embodiments, the dose of modified cells is administered to a subject in need thereof in a single dose or multiple doses. In some embodiments, the dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every three weeks or every 21 days, once every 4 weeks or every 28 days. In exemplary embodiments, a single dose of modified cells is administered to a subject in need thereof. In exemplary embodiments, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cell or recombinant receptor, the severity and course of the disease, whether the cell is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history and response to the cell of the subject, and the discretion of the attending physician. In some embodiments, the compositions and cells are suitably administered to a subject at one time or within a series of treatments.

In some embodiments, the cells are administered as part of a combination therapy, such as simultaneously or sequentially, or in any order, with another therapeutic intervention, e.g., an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. In some embodiments, the cells are co-administered with one or more additional therapeutic agents or are administered simultaneously or sequentially in any order with another therapeutic intervention. In some cases, the cells are co-administered with another therapy at a time sufficiently close that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the method comprises administering a chemotherapeutic agent.

In some embodiments, after administration of the cells, the biological activity of the engineered cell population is measured, for example by any of a number of known methods. Parameters to be assessed include specific binding of engineered or native T cells or other immune cells to the antigen, in vivo, such as by imaging, or ex vivo, such as by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as, for example, the Methods described in Kochenderfer et al, j.immunothergy, 32(7):689-702(2009), and Herman et al j.immunological Methods,285(1):25-40 (2004). In certain embodiments, the biological activity of a cell is measured by determining the expression and/or secretion of one or more cytokines, such as CD107a, IFNy, IL-2, and TNF. In some aspects, biological activity is measured by assessing clinical outcome (such as a reduction in tumor burden or burden).

In some embodiments, particular dosage regimens of the present disclosure include a lymphocyte clearance (lymphodepletion) step prior to administration of the modified T cells. In an exemplary embodiment, the lymphocyte depleting step comprises administering cyclophosphamide and/or fludarabine.

In some embodiments, the lymphocyte depleting step comprises administering cyclophosphamide at a dose of: about 200mg/m²Daily and about 2000mg/m²Between one day (e.g., 200 mg/m)²300 mg/m/day²One day, or 500mg/m²Day). In an exemplary embodiment, the dose of cyclophosphamide is about 300mg/m²The day is. In some embodiments, the lymphocyte depleting step comprises administering fludarabine at a dose of: about 20mg/m²Daily and about 900mg/m²Between one day (e.g., 20 mg/m)²25 mg/m/day ²30 mg/m/day²One day, or 60mg/m²Day). In an exemplary embodiment, the dose of fludarabine is about 30mg/m²The day is.

In some embodiments, the lymphocyte depleting step comprises administering cyclophosphamide at a dose of: about 200mg/m²Daily and about 2000mg/m²Between one day (e.g., 200 mg/m)²300 mg/m/day²One day, or 500mg/m²Day), and fludarabine was administered at the following dose: about 20mg/m²Daily and about 900mg/m²Between one day (e.g., 20 mg/m)²25 mg/m/day ²30 mg/m/day²One day, or 60mg/m²Day). In an exemplary embodiment, the lymphocyte clearance step comprises a treatment at about 300mg/m²Cyclophosphamide is administered at a daily dose and at about 30mg/m²Fludarabine was administered at a dose per day.

In an exemplary embodiment, for a subject with castration-resistant prostate cancer, the subject receives lymphodepleting chemotherapy prior to administration of the modified T cells. In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives an intravenous infusion comprising equal to or about 500mg/m²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for a subject with castration-resistant prostate cancer, the subject receives an intravenous infusion comprising equal to or about 500mg/m approximately 3 days (+ -1 day) prior to administration of the modified T cells²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for patientsA subject having castration-resistant prostate cancer, the subject receiving an intravenous infusion comprising equal to or about 500mg/m up to 4 days prior to administration of the modified T cells²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives an intravenous infusion comprising equal to or about 500mg/m 4 days prior to administration of the modified T cells ²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives an intravenous infusion comprising equal to or about 500mg/m 3 days prior to administration of the modified T cells²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for a subject with castration resistant prostate cancer, 2 days prior to administration of the modified T cells, the subject receives an intravenous infusion comprising equal to or about 500mg/m²To equal to or about 1g/m²Is a lymphodepleting chemotherapy of cyclophosphamide.

In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives an intravenous infusion comprising 300mg/m 3 days prior to administration of the modified T cells²Is a lymphodepleting chemotherapy of cyclophosphamide. In an exemplary embodiment, for a subject with castration resistant prostate cancer, prior to administration of the modified T cells, the subject receives a composition comprising 300mg/m by intravenous infusion²The lymphodepleting chemotherapy of cyclophosphamide continues for 3 days.

In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives a composition comprising about 20mg/m²Daily and about 900mg/m²Between one day (e.g., 20 mg/m)²25 mg/m/day ²30 mg/m/day²One day, or 60mg/m²Day) of fludarabine. In an exemplary embodiment, the subject having castration-resistant prostate cancer is treated withThe subjects received a dose of 30mg/m²The dose of fludarabine was administered for 3 days.

In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives a composition comprising about 200mg/m²Daily and about 2000mg/m²Between one day (e.g., 200 mg/m)²300 mg/m/day²One day, or 500mg/m²A day) of cyclophosphamide and about 20mg/m²Daily and about 900mg/m²Between one day (e.g., 20 mg/m)²25 mg/m/day ²30 mg/m/day²One day, or 60mg/m²Day) of fludarabine. In an exemplary embodiment, for a subject with castration resistant prostate cancer, the subject receives a composition comprising about 300mg/m²Cyclophosphamide in a daily dose and 30mg/m²The lymphodepleting chemotherapy of fludarabine (lefludarabine) continued for 3 days.

It is known in the art that one adverse effect following infusion of CAR T cells is the onset of immune activation, known as Cytokine Release Syndrome (CRS). CRS is an immune activation that leads to an increase in inflammatory cytokines. Clinical and laboratory measurements range from mild CRS (systemic symptoms and/or grade 2 organ toxicity) to severe CRS (sCRS; > grade 3 organ toxicity, aggressive clinical intervention, and/or possible life-threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia/hypotension, capillary leakage, cardiac dysfunction, kidney damage, liver failure and extensive intravascular coagulation. Following CAR T cell infusion, a dramatic increase in cytokines has been shown, including interferon- γ, granulocyte macrophage colony stimulating factor, IL-10, and IL-6. The presence of CRS is often associated with the expansion and progressive immune activation of adoptively transferred cells. CRS severity has been shown to be determined by disease burden at the time of infusion, as patients with high tumor burden experience more CRS.

Thus, the present invention provides appropriate CRS management strategies following CRS diagnosis to alleviate the physiological symptoms of uncontrolled inflammation without inhibiting the anti-tumor efficacy of engineered cells (such as CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse the symptoms of CRS (e.g., grade 3 CRS) without compromising the initial anti-tumor response.

In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the food and drug administration approved monoclonal antibody tocilizumab, also known as amituzumab (atlizumab) (sold as actetrara or RoActemra). Tolizumab is a humanized monoclonal antibody directed against the interleukin-6 receptor (IL-6R). Administration of tollizumab has been shown to reverse CRS almost immediately.

In some embodiments, the methods of the invention involve selecting and treating a subject who has failed in at least one prior course of therapy for the cancer. For example, a suitable subject may have been diagnosed with recurrent prostate cancer. In some embodiments, the methods of the invention involve selecting and treating a subject who has received a standard prior course of therapy for at least one cancer. For example, a suitable subject may have previously been treated for metastatic castration-resistant prostate cancer with at least one standard 17 α lyase inhibitor or a second generation anti-androgen therapy.

In an exemplary embodiment, a suitable subject is a subject having metastatic castration-resistant prostate cancer. In exemplary embodiments, a suitable subject is a subject with metastatic castration resistant prostate cancer that has ≧ 10% PSMA-expressing tumor cells as evidenced by immunohistochemical analysis of fresh tissue.

In some embodiments, a suitable subject is one with radiographic evidence of bone metastatic disease and/or measurable non-bone metastatic disease (lymph nodes or internal organs).

In some embodiments, a suitable subject is a subject with an ECOG performance status of 0-1.

In some embodiments, a suitable subject is a subject with sufficient organ function, as defined below: serum creatinine is less than or equal to 1.5mg/dl or creatinine clearance is more than or equal to 60 cc/min; and/or serum total bilirubin <1.5x ULN; serum ALT/AST <2x ULN.

In some embodiments, a suitable subject is a subject with sufficient hematological characteristics, as defined below: hgb is greater than 10 g/dl; PLT >100 k/ul; and/or ANC >1.5 k/ul.

In some embodiments, suitable subjects are non-infusion dependent subjects.

In some embodiments, a suitable subject is a subject with evidence of progressive castration-resistant prostate adenocarcinoma, defined as follows: castration levels of testosterone with or without androgen deprivation therapy (<50 ng/ml); and/or evidence of one of the following measures of progressive disease: with progression of bone disease with 2 or more new lesions on bone scans according to RECIST1.1 standard soft tissue progression (as per PCWG2 standard), serum PSA increased by at least 25% and an absolute increase of 2ng/ml or more from nadir (as per PCWG2 standard).

In some embodiments, a suitable subject has been previously treated with at least one second-generation androgen signaling inhibitor. In some embodiments, a suitable subject has been previously treated with abiraterone (abiraterone). In some embodiments, a suitable subject has been previously treated with enzalutamide (enzalutamide).

In some embodiments, a suitable subject has ≧ 10% PSMA-expressing tumor cells found on metastatic tissue biopsy by Immunohistochemistry (IHC).

In some embodiments, suitable subjects have radiographic evidence of metastatic disease (bone or lymph node/internal organ).

In some embodiments, suitable subjects have a line of therapy (lines of therapy) of ≦ 4 for metastatic CRPC.

K. Pharmaceutical compositions and formulations

Also provided are populations of immune cells of the invention, compositions containing such cells and/or enriched for such cells, such as where the cells expressing the recombinant receptor comprise at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells within the composition, or of a certain type of cell, such as a T cell or CD8+ or CD4+ cell. Among these compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are methods of treatment that administer the cells and compositions to a subject (e.g., a patient).

Also provided are compositions including cells for administration, including pharmaceutical compositions and formulations, such as unit dosage compositions, that include the number of cells administered in a given dose or portion thereof. Pharmaceutical compositions and formulations typically include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one additional therapeutic agent.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain additional ingredients that would cause unacceptable toxicity to the subject to which the formulation is administered. By "pharmaceutically acceptable carrier" is meant an ingredient of a pharmaceutical formulation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the selection of the vector is determined in part by the particular cell and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by total weight of the composition. Vectors are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, phenol, butyl or benzyl alcohol, parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; classes such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions (salt-forming counter-ion), such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG).

In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by total weight of the total composition. Methods of preparing administrable pharmaceutical compositions are known. In the fields of, for example, Remington: Science and Practice of Pharmacy, Lippincott Williams & Wilkins; exemplary methods are described in more detail in 21 st edition (5/1/2005).

The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient for the particular indication, disease or condition being treated with the cells, preferably those having activities complementary to the cells, wherein the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin (daunorubicin), doxorubicin (doxorubicin), fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab (rituximab), vinblastine, and/or vincristine. In some embodiments, the pharmaceutical composition contains an amount of cells effective to treat or prevent a disease or disorder, such as a therapeutically effective amount or a prophylactically effective amount. In certain embodiments, therapeutic or prophylactic efficacy is monitored by periodic assessment of the subject being treated. The desired dose may be delivered by administering the cells as a single bolus, by administering the cells as multiple boluses, or by administering the cells as a continuous infusion.

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, the cells are administered to the subject by peri-systemic delivery, such as intravenous, intraperitoneal, or subcutaneous injection. In some embodiments, the compositions are provided as sterile liquid formulations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, the viscous composition may be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. The liquid or viscous composition can comprise a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent or excipient (e.g., sterile water, saline, glucose, dextrose, and the like). The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring and/or coloring agents, depending on the route of administration and the desired formulation. In certain aspects, suitable formulations can be prepared with reference to standard text.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, can be used to ensure protection against the action of microorganisms. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Formulations for in vivo administration are typically sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information in any such articles, patents, patent applications, or other physical or electronic documents.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Other suitable modifications and variations to the methods described herein may be made by those skilled in the art using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to fall within the scope of the appended claims. Having now described certain embodiments in detail, these embodiments will be more clearly understood by reference to the following examples, which are intended for purposes of illustration and are not intended to be limiting.

Examples

The invention will now be described with reference to the following examples. These examples are provided for illustrative purposes only, and the present invention is not limited to these examples, but encompasses all variations apparent from the teachings provided herein.

The materials and methods employed in these experiments are now described.

RNA CAR construct design: four human scFvs specifically targeting human PSMA were synthesized by IDT: 1C3, 2A10, 2C6 and 2F5 as gBlocks. The CAR with 4-1 BB-zeta (BBZ) was assembled by overlap PCR and cloned into the RNA in vitro transcription vector pD-A. The pD-A vector was optimized for T cell transfection, CAR expression and RNA production. Prior to RNA IVT, four human PSMA CARs and one mouse PSMA CAR (J591) were linearized by SpeI digestion. The T7mScript Standard mRNA production System (Cellscript, Inc., Madison, Wis.) was used to generate capped/tailed IVT RNAs. IVT RNA was purified by RNeasy Mini Kit (Qiagen, Inc., Valencia, Calif.). Purified RNA was eluted at 1-2 mg/mL in RNase-free water and stored at-80 ℃ until use. The integrity of the RNA was confirmed by 260/280 absorbance and visual inspection on an agarose gel.

Lenti CAR construct design: all PSMA CARs were subcloned into pTRPE Lenti vector. The receptor will then be switched: PD1, CD28-F2A (SW), PD1^A132Lcd28-F2A (SW) and the dominant negative TGFR β II sequence, nTGFR β II-T2A (dn) were subcloned into each Lenti vector, followed by human PSMA scFv.

Examples of sequences included in the Lenti vector are as follows:

1C3(SEQ ID NO:169)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAACAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGTCCCCTGGGGATCGAGGTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCTCCTAAGCTCCTGATCTTTGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAACAGTTATCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCG

2A10(SEQ ID NO:170)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGGCAAACTGGTTTCCTCTGGTCCTCCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCTATGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAACCACGACGCCAGCGCCGCG

2F5(SEQ ID NO:171)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGTTTTACCAGCAACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAACAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAACTGGTTTCCTCTGGTCCTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGACATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCGGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAATCAAAACCACGACGCCAGCGCCGCG

2C6(SEQ ID NO:172)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAGCTGGTGCAGTCTGGATCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAACTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTATCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTCCCGGGTATACCAGCAGTTGGACTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCCCTATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAACCACGACGCCAGCGCCGCG

PD1.CD28-F2A(SW)(SEQ ID NO:173)

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG

PD1^A132L-PTM.CD28-F2A(SW*)(SEQ ID NO:174)

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGCTGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGTTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGCTAGTCTGGGTCCTGGCCGTCATCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCG

dnTGFRβII-T2A(dn)(SEQ ID NO:175)

ATGGGTCGGGGGCTGCTCAGGGGCCTGTGGCCGCTGCACATCGTCCTGTGGACGCGTATCGCCAGCACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACTTGTTGCTAGTCATATTTCAAGTGACAGGCATCAGCCTCCTGCCACCACTGGGAGTTGCCATATCTGTCATCATCATCTTCTACTGCTACCGCGTTAACCGGCAGCAGAAGCTGAGTTCATCCGGAAGATCTGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGAGCCACC

Transduction protocol: a large number of T cells (CD4 and CD8) obtained from Human Immunology Core were diluted to 10⁶cells/mL and stimulated with CD3/28 beads (T cell expansion agent, Invitrogen) at a cell to bead ratio of 1: 3. Transduction of packaged lentiviral vectors with a MOI of 3:1 at 1 day post stimulation and allowed to proceed at 37 ℃/5% CO₂And (5) amplification in an incubator.

Transduction efficacy: CAR transduction efficacy was assessed by flow cytometry using either Biotin-SP-AffiniP goat anti-mouse IgG (Cat #: 115-. APC anti-human CD279(PD-1) antibody (Cat #:329908, BioLegend) and human TGF-. beta.RII APC-conjugated antibody (Catalog # FAB241A, R & D systems) were used to examine the switch receptor or dominant negative TGFR. beta.II moiety.

T cell expansion: cells were fed and divided every 2 days starting on day 3 post stimulation. T cells were debeaded on day 4 and frozen on day 10 for later use.

RNA electroporation: dormant T cells were electroporated with 10 or 20 μ g IVT PSMA RNA CAR using BTX830 at 500V and 700 μ s. Nalm6.CBG or K562 cells were electroporated with 5. mu.g or 15. mu.g PSMA IVT RNA at 300V and 500. mu.s using BTX 830. PC3.PSMA cells were electroporated with 0.5. mu.g, 2. mu.g, or 5. mu.g PDL1IVT RNA using BTX830 at 300V and 500. mu.s. Immediately after electroporation, cells were placed at 37 ℃ and 5% CO ₂In the pre-warmed culture of (4). After 18 hours, PSMA or PDL1 electroporated tumor cells were stained with either an APC anti-human PSMA (FOLH1) antibody (Cat #: 342507, BioLegend) or an APC anti-human CD274(PDL-1 or B7-H1) antibody (Cat #: 17-5983-42, BD Biosciences) and analyzed by flow cytometry.

Cell counting: at various time points during the expansion-resting cycle, cells were gently mixed and 40 μ L aliquots of cells were collected from known culture volumes and placed in cuvettes (Beckman Coulter) with 20mL Isoton II diluent buffer for counting using a Coulter Multisizer 3(Beckman Coulter) according to CCI laboratory SOP. These measurements determine cell concentration, total cell number, growth rate, and cell volume, and are used to calculate dilution volume and determine when cells are dormant for freezing.

quantitative-PCR: primary cells or tumor cell lines were lysed and passed through a QIA disruptor (Cat # 79656). Total RNA was extracted by RNeasy Mini kit (Cat #74104) according to the manufacturer's protocol. Reverse transcription was performed (Cat #:11904-018, Invitrogen) to obtain cDNA. Quantitative PCR of cDNA with primers specific for PSMA or GAPDH: PSMA (F primer: AGGAAGTCTCAAAGTGCCCT (SEQ ID NO:176), R primer: GAACAACAGCTGCTCCACTC (SEQ ID NO:177)) or GAPDH (F primer: GCTACACTGAGCACCAGGTGGTCTC (SEQ ID NO:178), R primer: CCCAGCAGTGAGGGTCTCTCTCTTC (SEQ ID NO: 179)).

ELISA for IL-2 and IFN-. gamma.: t cells or target cells were washed and washed at 1X10⁶cells/mL were suspended in R10 medium. Approximately 0.1mL of each cell line was added to the wells of a 96-well plate (Corning) and incubated at 37 ℃ for 18 to 20 hours. Supernatants were harvested and ELISA performed.

CD107a assay: preparation of E in 100 μ L of R10 medium: t ratio of 1:2 (5X 10)⁴Effector 1X10⁵Target) and plated into 96-well plates. Add 10. mu.L phycoerythrin-labeled anti-CD 107a Ab and incubate the plates at 37 ℃ for 1 hour. Golgi Stop (2 ul Golgi Stop in 3ml R10 medium, 10 ul/well; BD Biosciences, 51-2092KZ) was added and the plates were incubated for an additional 2.5 hours. Then, 2. mu.L of FITC-anti-CD 8(Cat #: 551347, BD Pharmingen) and 2. mu.L of APC-anti-CD 3(Cat #: 555342, BD Pharmingen) were added and incubated at 37 ℃ for 30 minutes. After incubation, the samples were washed with FACS buffer and analyzed by flow cytometry.

Luciferase-based CTL assay: nalm6-CBG, PC3-CBG, PC3.PSMA-CBG tumor cells were cultured in R10 medium at 1X10⁵The individual cells/mL are resuspended and incubated with different ratios of T cells (e.g., 10:1, 5:1, 2.5, etc.) at 37 ℃ for 18 hours. Equal volumes of substrate were added and the luminosity was immediately determined. Results are reported as based on the percent killing of luciferase activity in wells with tumor only in the absence of T cells (% killing 100- ((RLU from wells with effector and target cell co-culture)/(RLU from wells with target cell) × 100)).

Psma tumor model: 2E6 pc3.psma.7sc cells transduced with click beetles were injected into mice (i.v.) and 28 days later, 2E6 PSMA CAR-T positive transduced T cells were injected into tumor bearing mice (i.v.). Bioluminescence imaging (BLI) was performed at multiple time points.

The results of the experiment are now described.

Example 1: human RNA PSMA CAR has antitumor activity equivalent to mouse RNA PSMA CAR, J591

Four human RNA CAR targeting PSMA were constructed using one of the four scFv sequences (1C3(SEQ ID NO:169), 2A10(SEQ ID NO:170), 2C6(SEQ ID NO:172), and 2F5(SEQ ID NO:171) (from U.S. patent application US2009/0297438A1, the contents of which are incorporated herein by reference in their entirety)). The ScFv was linked to the CD8 transmembrane domain and the 4-1BB and CD3 zeta intracellular signaling domains. Purified RNA was visualized on agarose gel (fig. 1A) and electroporated into dormant human primary T cells. Under the conditions tested, all CARs had close to 100% CAR expression. 10 μ g IVT RNA CAR reached the maximum Mean Fluorescence Intensity (MFI) for CAR expression, whether it was human or mouse PSMA CAR; therefore, 10. mu.g of IVT RNA was used for further experiments (FIG. 1B). CAR expression varied among different human CARs, with the highest MFI in CAR expression being 1c3. bbz. Mouse J591 CAR expresses MFI 4-fold higher than 1c3. bbz; however, due to the different kinds of antibody sources, 10 μ g of mouse J591 RNA CAR was also used for further experiments.

Full-length PSMA was cloned into PD-a vector for optimal RNA expression. Purified RNA was visualized on agarose gel (fig. 1A), electroporated into nalm6.cbg or K562 tumor cells, and analyzed for PSMA expression by flow cytometry (fig. 1C). Further experiments were performed with 15 μ g and 5 μ g PSMA RNA for nalm6.cbg and K562 tumor cells, respectively. Previously constructed pc3.PSMA. psca. cbg tumor cells had reduced PSMA expression (37.5%) (fig. 1C). The pc3.psma.cbg tumor cell line was subjected to limiting dilution and 7 single cell clones were isolated and simultaneously combined into a new cell line, pc3.psma.7sc.cbg (fig. 1D).

Nalm6.cbg or K562 electroporated with PSMA RNA, PC3 or pc3.PSMA. psca. cbg tumor cells were co-cultured with various PSMA RNA CAR. CD107a assays, luciferase-based CTL assays, and ELISA assays were performed to determine the functionality of the four new human CARs. All four human PSMA CARs had degranulation activity equivalent to mouse PSMA CAR, J591, when co-cultured with PSMA positive cells (fig. 2A). However, three of the four human PSMA CARs exerted higher non-specific activation of PC3 cells than the J591 CAR (fig. 2A). All four human CARs had comparable cytotoxic and anti-tumor activity against PSMA positive cells compared to J591 CARs (fig. 2B and 2C). Cytokine production was a PSMA target specific for 1C3.bbz, 2a10.bbz and 2f5.bbzcar (fig. 2C).

Example 2: human Lenti PSMA CAR-specific targeting PSMA-positive cells

Four human PSMA CARs were subcloned into pTRPE Lenti vector. Primary human T cells transduced with human PSMA CARs had different CAR expression levels: 40% for 1c3.bbz, 66% for 2a10.bbz, 50% for 2c6.bbz, and 6% for 2f5.bbz (fig. 3A). The dominant negative TGFR β II sequence was linked to the mouse j591.bbz CAR by T2A sequence (dnTGFR β II-j591. bbz). Nalm6.cbg electroporated with PSMA RNA, PC3, or pc3.pcma. cbg cells were co-cultured with various PSMA Lenti CARs. CD107a assays, luciferase-based CTL assays, and ELISA assays were performed to determine the functionality of four new human PSMA Lenti CARs and compared to mouse J591 CARs. 1C3.BBZ, 2A10.BBZ and 2F5.BBZ exert similar antitumor activity as dnTGFR β II-J591 in degranulation assays and luciferase-based killing assays (FIG. 3B and FIG. 3C). For cytokine production, all four human PSMA Lenti CARs elicited specific IL-2 and INF- γ production, but the amounts varied in human CARs (fig. 3D).

Example 3: construction of conversion receptor or dominant negative-TGFR beta II linked-human LentiCAR

A switch receptor (pd1.cd28) comprising a truncated extracellular domain of PD1 and transmembrane and cytoplasmic signaling domains of CD28 was designed and linked to each human PSMA CAR via a T2A sequence. A point mutation from alanine to leucine at position 132 (mouse 99) on PD1 increased its affinity for PDL1 by a factor of 2 (Zhang et al. immunity 20, 337-347, 2004). Thus, the second form of the conversion receptor "PD 1 ^A132LCd28 "(with truncated extracellular and transmembrane domains of PD1 and cytoplasmic signaling domain of CD 28) was linked to each human PSMA CAR. The dominant negative TGFR β II sequence was also subcloned into each human PSMA CAR. Flow cytometry was performed to examine the transduction efficiency of each of the transduction receptors-CAR (fig. 4A) and dnTGFR β II CAR (fig. 4B). For all switch receptor-CAR transduced T cells, a distinct CAR/PD1 double positive population was observed. Transduction efficiencies were similar between human PSMA Lenti CARs and their two transducin receptor counterparts (counterparts) (fig. 4A). Each 2C6 CAR hasThe lowest transduction efficiency. For each dnTGFR β II-linked human PSMA CAR, there was no single dnTGFR β II population, but a significant shift was observed (fig. 4B).

To examine the function of transducible receptors, various amounts of PDL1 RNA (0.5ug, 2ug, and 5ug) were electroporated into pc3.psma cells (fig. 4C). PSMA cells electroporated with 5ug PDL1 RNA were co-cultured with each PSMA CAR. Before the co-culture experiment, dnTGFR β II-J591.BBZ was normalized to 50% transduction efficiency. Three of the four human PSMA CARs and their transducin receptor or dnTGFR β II counterparts showed comparable degranulation activity to dnTGFR β II-j591.bbz CAR when co-cultured with PSMA positive cells (fig. 4D, fig. 4E, and fig. 4F). Bbzcar and its related counterparts expressed lower degranulation activity, probably due to lower transduction efficiency (fig. 4G). All four human PSMA CARs and their switch receptors or dnTGFR β II counterparts showed similar cytotoxicity to pc3.PSMA cells (fig. 4H). All human PSMA CARs elicited cytokine equivalent amounts to dnTGFR β II-j591.bbz CARs (fig. 4I). Psma cells when co-cultured with PDL1 electroporated pc3. each transducing receptor CAR secreted almost two-fold more IL-2 than its non-transducing receptor or dnTGFR β II CAR counterpart (fig. 4I).

Example 4: PSMA.7SC xenograft model

The following six human PSMA CARs were selected for the pc3.psma.7sc mouse xenograft model: 1C3.BBZ, PD1CD28.1C3.BBZ, 2A10.BBZ, PD1CD28.2A10.BBZ, dnTGFR beta II-2A10.BBZ and dnTGFR beta II-J591. BBZ. CAR expression was tested by flow cytometry (fig. 5A and 5B). Mouse j591.bbz Lenti CAR was included in the functional assay. All PSMA CARs tested showed similar degranulation activity in the CD107a assay (fig. 5C and 5D) and comparable killing activity in the luciferase-based CTL assay, with 2a10.bbz lowest and dnTGFR β II-j591.bbz highest (fig. 5E). Each transducing receptor CAR secreted almost two-fold more IL-2 when co-cultured with pc3.psma.7sc cells electroporated with PDL1 compared to its non-transducing receptor or dnTGFR β IICAR counterpart (fig. 5F).

To ensure safety using the human PSMA CAR described above, a panel of primary human cells (table 1) were tested for PSMA expression by quantitative PCR (fig. 5G). All primary cells tested had different PSMA expression levels after normalization by nalm6.cbg, even PC3 cells, which had only limited reactivity to PSMA RNA CAR (fig. 2A) but not PSMA Lenti CAR (fig. 5H) (pc3.PSMA was 1800-fold higher than PSMA expression of PC3 cells). HREpC, HSAEpC and HPMEC human primary cells were co-cultured with the above CARs. CD107a and ELISA assays were performed. All tested PSMA CARs had minimal detectable degranulation activity when co-cultured with HPMEC (fig. 5H and fig. 5I). Pd1cd28.1c3.bbz caused an increase in IL-2 compared to 1c3.bbz when co-cultured with HPMEC (fig. 5J). Although primary human cells elicited cytokine levels, particularly HPMEC, negligible compared to those elicited by pc3.psma.7sc (fig. 5J compared to fig. 5F).

Table 1: primary human cells for testing PSMA expression

HRCEpC	Human renal cortical epithelial cells
		Hn2	Primary human neurons
hNP1	Human neuron progenitor cell
		hMSC-BM	Human mesenchymal stem cells from bone marrow
HPASMC	Human pulmonary artery smooth muscle cells
		HCM	Human cardiac muscle cell
HOB	Human osteoblast
		HAoSMC	Human aortic smooth muscle cells
HREpC	Human renal epithelial cells
		HPAEC	Human pulmonary artery endothelial cells
Kera	Keratinocyte (Keratinocyte)
		HSAEpC	Human small airway epithelial cells
HPMEC	Human lung microvascular endothelial cells

In vivo NSG mice experiment 7 groups (5 mice per group) were designed to test the above 6 PSMA CARs plus an untransduced control group. 2E6 pc3.psma.7sc cells transduced with green click beetles were injected into mice (i.v.) and after 28 days 2E6CAR positive transduced T cells were injected into tumor bearing mice (i.v.). Bioluminescence imaging (BLI) was performed at different time points: day 27, 34, 42, 49 post tumor injection (fig. 5K and fig. 5L). Without being bound by any theory, the results of this experiment show that all PSMA CARs tested have anti-tumor activity comparable to dnTGFR β ii.j591.bbz.

FIG. 6 shows the different domains of the dnTGFRII-T2A PSMA-CAR construct, as well as the pTRPE dnTGFRII-T2A PSMA CAR vector map.

Example 5: in vivo tumor control by PSMA CAR-T cells

Transduction protocol: a large number of T cells (CD4 and CD8) obtained from Human Immunology Core were diluted to 10⁶Individual cells/mL and with CD3/28 beads (T cell expansion agent, Invitrogen) at a 1:3 cell: the beads were stimulated. Transduction of packaged lentiviral vectors with a MOI of 3:1 at 1 day post stimulation and allowed to proceed at 37 ℃/5% CO₂And (5) amplification in an incubator.

Transduction efficacy: transduction efficacy was assessed by flow cytometry using PE anti-human TCR ν β 8 antibody (Cat #:348104, BioLegend) and APC anti-human CD279(PD-1) antibody (Cat #:329908, BioLegend).

T cell expansion: cells were fed and divided every 2 days starting on day 3 post stimulation. T cells were debeaded on day 3 or day 4 and frozen on day 12 for later use.

Cell counting: at various time points during the expansion-resting cycle, cells were gently mixed and 40 μ L aliquots of cells were collected from known culture volumes and placed in cuvettes (Beckman Coulter) with 20mL Isoton II diluent buffer for counting using a Coulter Multisizer 3(Beckman Coulter) according to CCI laboratory SOP. These measurements can determine cell concentration, total cell number, growth rate, and cell volume, and are used to calculate dilution volume and determine when cells are dormant for freezing.

ELISA for IL-2 and IFN-. gamma.: t cells were washed and washed at 1x10⁶cells/mL were suspended in R10 medium. Approximately 0.1mL of each cell line was added to the wells of a 96-well plate (Corning) and incubated at 37 ℃ for 18 to 20 hours. Supernatants were harvested and ELISA performed.

CD107a assay: target (E: T) cell ratio of 1:1 effector to target (E: T) in 96-well plates containing 160. mu. L R/10 medium (10)⁵ Effector 10⁵Target) seeded cells. anti-CD 107a was added and incubated with the cells at 37 ℃ for 1 hour, then Golgi Stop was added and incubated for another 2.5 hours. anti-CD 8 and anti-CD 3 antibodies were then added and incubatedIncubate at 37 ℃ for 30 minutes. After incubation, the samples were washed once and analyzed by flow cytometry using BD Accuri C6. Data was analyzed using FlowJo software.

PC3-PMSA tumor model: 1E6 PC3-PMSA-CBG was injected subcutaneously (s.c.) into mice, and after 21 days, lentivirus transduced T cells were injected intravenously (i.v.) into tumor-bearing mice. Bioluminescent imaging (BLI) and tumor measurements were performed at multiple time points.

As a result: the sequences described in table 2 were generated and tested for their ability to control tumors in vivo.

Table 2: PSMA CAR in combination with various switch receptor sequences

PSMA CARs with ICOS or combinations of ICOS. ymnm signaling domains and CAR + PD1 (or Tim3) switch receptors were constructed and cloned into lentiviral vectors (see, sequences of table 2). CAR expression levels in transduced T cells were comparable for most CAR constructs (figure 7), and the transduction receptors were moderately expressed (figure 8). PSMA CARs with icos. ymnm signaling domain (ICOSymnm) showed significantly higher CD107a expression compared to wild-type icos (icos) or 4-1BB (41BB) CARs when stimulated with PSMA positive cell line pc3.PSMA or pc3.PSMA. pd-L1 and examined for CD107a upregulation (figure 9). ICOS and ICOS. ymnm PSMA CAR granzyme B expression was similar to 4-1BB PSMA CAR (fig. 10). Cytokine production (IL-2 and IFN- γ) was significantly higher for PD1 switch receptors co-transduced with PSMA CARs including ICOS, ICOS. ymnm, or 4-1BB when stimulated with PD-L1 expressing pc3.PSMA cells (fig. 11A and 11B).

Fig. 12 shows the quantitative results of bioluminescence imaging for NSG mice bearing PC3-psma. cbg-induced tumors treated with indicated CAR-transduced T cells for up to 86 days (fig. 12A), and up to 151 days (fig. 12B). Figure 13 shows NSG mice bearing PC3-psma. cbg-induced tumors treated with indicated CAR-transduced T cells for up to 164 days of tumor size. As shown, ICOS (2f5.icosz) and ICOS. ymnm PSMA CAR (2f5.icoszymnm) both showed poorer tumor control than 4-1BB PSMA CAR (2f5. bbz). The PD1-CD28 switch receptor improved PSMA CARs with the 4-1BB co-stimulatory domain (pd1.cd28.2f5.bbz), but not ICOS (pd1cd28.2f5icosz) or ICOS. ymnm (pd1cd28.2f5icoszymnm). Both pd1.cd28.2f5.bbz and pd1cd28.2f5icoszymnm showed poor tumor control compared to 4-1BB PMSA CAR. When these ICOS-based CARs were co-delivered to T cells with a high affinity PD1 switch receptor with a 4-1BB signaling domain (PD1 × BB), tumors could be controlled as effectively as 4-1BB PMSA CARs. As shown in figures 14A to 14F, co-delivery of T cells with icos.ymnm CAR and PD1 switch receptor with 4-1BB signaling domain (PD1 bb.2f5icoszymnm) abolished the tumor. Fig. 14G provides a list of T cells in order of tumor control ability.

Example 7: PSMA CAR-T cells co-express bispecific that switch PD1 to CD28 or TGF beta receptor II to CD28 Specific antibodies

Five bispecific antibodies that can bind PD-L1(10A5, 13G4 and 1B12, see, e.g., PCT publication No. WO2007005874A2) or the scFC of TGF β receptor II (aTGFbRII-1 and aTGFbRII-3(TGFb1 and TGFb3, see, e.g., U.S. Pat. No. 8,147,834) and anti-CD 28 scFv (1412, see, e.g., U.S. Pat. No. 7,585,960) are designed for use and genes are synthesized by PCR.

Generating a PSMA CAR-T cell that co-expresses a bispecific antibody selected from those described above.

Example 8: manufacture and administration of clinical CART-PSMA-TGF beta RDN autologous T cells

CART-PSMA-TGF β RDN research cell product manufacture, final formulation, testing and labeling were performed as described below according to the clinical Cell and Vaccine Production Facility (CVPF) Standard Operating Protocol (SOP). CVPF is an individual within the department of transfusion medicine and treatment pathology at the university of pennsylvania department of pathology and laboratory medicine. In this sector, in addition to CVPF and apheresis, there is a separate hematopoietic stem cell processing laboratory responsible for bone marrow and peripheral blood stem cell production, primarily dedicated to support clinical hematopoietic stem cell transplantation services. CVPF is a registered HCT facility and has been certified by the cell therapy certification institute (FACT).

Dynabeads CD3/C28 CTS^TM(formerly called ClinExVivo) beads were used for T cell activation and expansion.

CART-PSMA-TGF β RDN research product manufacture began with the apheresis product. The following occurs based on the composition of the apheresis product evaluated by the Beckman Coulter Multisizer and BD FACS Calibur apparatus: mononuclear cells were removed by counter-current centrifugal elutriation on TerumoBCT Elutra using a disposable closed system disposable set, a washing step using a semi-automated closed system set Haemonetics CellSaver 5, and/or Ficoll separation of the dark yellow (buffy) fraction of PBMCs. On day 0, the CART-PSMATGF β RDN manufacturing process was initiated by activating T lymphocytes with Dynabeads CD3/CD28 CTS beads. PSMA-TGFbRIIDN CAR LV vector was added at total final MOI on day 1. Vector transduction occurred between day 1 and day 3. On day 3, cells were washed and medium was changed. The cultures were allowed to continue to expand in the GE Wave bioreactor system. On the last day of culture, cells were harvested and concentrated using Cell Saver before harvest, and the Cell product was placed on Baxter MaxSep to remove Dynabeads CD3/CD28 CTS beads. After bead removal, the Cell amplificate is washed using Haemonetics Cell Saver 5 to remove residual vector, virus particles and Cell debris. CART-PSMA-TGF β RDN cells were resuspended in cryopreservation medium containing 31.25% plasma-a, 31.25% glucose 5%, 0.45% NaCl, 1 % dextran 40 and 5% glucose, 5% human serum albumin, and 7.5% DMSO. Cells were frozen in Cryostore vinyl acetate (EVA) (OriGen Biomedical) or equivalent clear bags using a controlled rate freezer.

Cryopreserved CART-PSMA-TGF β RDN: each infusion bag contained 10 to 50mL of cells. Cryopreserved cells were also retained in small aliquots of the same cell concentration as the infusion dose and used as sentry vials (sentinel visual) for viability and endotoxin testing prior to infusion and for stability testing.

Leukapheresis collection and cell separation/enrichment: autologous peripheral blood lymphocytes were collected by apheresis at the apheresis unit of the university of pennsylvania Hospital (HUP). Cryopreserved historical apheresis products collected from patients prior to study entry may be used for CART-PSMA-TGF β RDN cell manufacturing. If used, the sample must be collected at a properly certified apheresis center and the product must meet adequate monocyte yields.

Processing approximately 10-15L of blood on a COBE Spectra apheresis system or equivalent to obtain approximately 5X10⁹A population of individual white blood cells. In addition to the screening test requirements provided in the protocol, blood from all apheresis donors were tested for infectious disease by the national red cross test laboratory.

The single blood draw ingredient products were transported to the CVPF in an insulated container and the temperature was recorded at the time of receipt. Samples were taken for bacterial and fungal cultures, real-time phenotypic analysis by flow cytometry, and for research and related research purposes. The single blood collection liquid component product is stored under refrigeration or processed by elutriation. After panning or cell washing, cell number was determined on a Coulter Multisizer M3/M4 and viability was determined by trypan blue dye exclusion assay. The elutriated product is stored refrigerated or further processed. Cryopreserved apheresis or elutriation products are thawed and washed prior to culturing to remove the cryopreserved medium. These products were then processed by 1) washing and seeding with panning lymphocytes, 2) positive selection with CD3/CD28 beads, or 3) further T cell selection based on Ficoll gradient separation.

Culture initiation and amplification: using Dynabeads CD3/CD28 CT in static tissue culture flasksS stimulation of enriched lymphocytes to 8X10 in XVIVO-15 medium (modified X-VIVO15 medium) supplemented with 5% human AB serum, 2mM L-GlutaMAX, 20mM Hepes, 1mM sodium pyruvate, 1% MEM vitamin essential mix, 10mM N-acetylcysteine, and 100IU/ml IL-2 ⁵To 1x10⁶Suitable range of individual cells. Beads were added at a bead to cell 3:1 ratio. On day 5 of culture, if an acceptable cell number is reached, the cells are transferred to a WAVE2/10EH bioreactor to be expanded to the appropriate cell number for harvesting, electroporation, sampling and final formulation. On the last day of culture, cells were harvested and concentrated using the Cell Saver Wash system. Prior to harvest, the cell product was placed on Baxter MaxSep to remove anti-CD 3/CD28 magnetic microbeads. After harvesting, expanded T cells were plated at 2x10⁶Individual cells/mL were resuspended in X-VIVO medium supplemented with 5% human AB serum. The cells were placed in an incubator at 37 ℃ overnight.

CART-PSMA-TGFBRDN dosage formulation: for one group, the dosage formulations were at 1-3x10⁷/m²Dose initiation for CART-PSMA-TGFBRDN cells, and for other groups, dose formulations were at 1-3x10⁸/m²The dosage of (a) is started. Administration was based on anti-PSMA CAR expression. The total dose is formulated as a single dose.

Final formulation: after incubation, after removal of all release test samples and archives, cells were resuspended in insoluble cryopreservation medium containing 31.25% plasma cell-a, 31.25% dextrose (5%) in NaCl (0.45%), 7.5% DMSO, 5% human serum albumin, and 1% low molecular weight dextran (LMD).

Product administration:

cell thawing: cells were thawed by trained personnel at CVPF or bedside using a water bath or similar device maintained at 36 ℃ to 38 ℃. When the container is connected to an i.v. tube, no frozen block should be left in the container. If the CART-PSMA-TGF β RDN cell product exhibits a broken or leaky bag, or is otherwise compromised, then it is not infused and the CVPF is returned.

Application: infusion was performed in an isolation room of the CTRC or elsewhere at the university of pennsylvania hospital using prophylaxis of immunosuppressed patients. Prior to infusion, both individuals independently verified the information on the infusion product tag in the presence of the subject and confirmed that the information correctly matched the participants. Cells were infused within about 30 minutes after thawing. CART cells were infused intravenously into number 18 intravenous catheters via either the peripheral vein (preferred) or the central vein. CART cells were infused by gravity (i.e., without an infusion pump) through a latex free Y-type transfusion cuff with a 3-way stopcock using a Macrodrip intravenous tube at a rate of about-10 mL/min. Leukoreduction filters are not used for infusion of CART cell products. If the subject has an allergic reaction, or a severe hypotensive risk or any other reaction to the infusion, an emergency medical device (i.e., emergency cart) is available during the infusion. Vital signs (temperature, respiration rate, pulse, blood pressure and oxygen saturation by pulse oximeter) were measured before and after infusion. If the subject's vital signs are unsatisfactory and stable, the vital signs are continuously monitored for stability, at least every hour or clinical indication. After the physician administering the subject determines that the subject is in a satisfactory state, the subject may be discharged.

Example 9: CART-PSMA-TGF beta RDN clinical trial design

This protocol tested the safety of administering 2 dose levels of CART-PSMA-TGF β RDN cells intravenously after lymphodepletion with a moderate dose of cyclophosphamide administered alone or three days prior to CART-PSMA-TGF β RDN cells. Dose escalation follows a 3+3 design. CART-PSMA-TGF β RDN cells were permanently modified to target PSMA proteins with an anti-PSMA CAR fused to the signaling domain of 4-1BB and TCR ζ. The study population included patients with castration-resistant prostate cancer with radiographic evidence of lymph node, visceral or bone metastases. All patients had to progress after treatment with at least one standard 17 α lyase inhibitor or second generation antiandrogen therapy.

The first patient was dosed on a day of 2017, 8 months and 31 days.

As part of informed consent, subjects were asked to allow testing of their tumors for PSMA as one of the eligibility criteria. Preferably PSMA expression is assessed in fresh tumor biopsies; however, if the biopsy is not feasible or clinically inappropriate, archival tissue from the most recent metastatic tissue biopsy is used to determine if eligibility was obtained within the previous 90 days.

Eligible patients were confirmed to have PSMA expression in > 10% of tumor cells and met all other inclusion criteria.

Group 1 subjects (N-3 or 6) received a single dose of 1-3x10 on day 0⁷/m²Lentivirus-transduced CART-PSMA-TGF β RDN cells without any adjustment to the chemotherapy regimen. If the number of CAR T cells produced does not meet the predefined 1x10⁷/m²The minimum infusion dose of cells, no dose was administered and the subject was replaced in the study. If 1 DLT/3 subject is present, the study enrolled an additional 3 subjects at this dose level. If there were 0 DLT/3 subjects or 1 DLT/6 subjects, then the study progressed to group 2. If 2 DLT/3 subjects were at 1-3x10⁷/m²The dose of cells appeared, the enrollment of this group was stopped and the dose was reduced by 10-fold to 1-3x10⁶Cell/m²(group-1). In this case, a maximum of 6 subjects were enrolled in group-1.

Group 2 subjects (N-3 or 6) received a single dose of 1-3x10 on day 0⁸/m²Lentivirus-transduced CART-PSMA-TGF β RDN cells without any adjustment to the chemotherapy regimen. If the number of CAR T cells produced does not meet the minimum 1x10 specified by the protocol⁸/m²Cells, but satisfying at least 1x10 ⁷/m²The subject received the dose but was not included in the DLT assessment of group 2. The subject will be replaced with DLT assessment at this dose. If the number of CAR T cells manufactured does not meet the pre-specified dose, as outlined in group 1, no dose was administered and the subject was replaced in the study. If 1 DLT/3 subject is present, then the study enrolls 3 additional subjects at this dose level. If there are 0 DLT/3 subjects or 1 DLT/6 subjects, the study progresses to the groupAnd (3) otherwise. If 2 DLT/3 subjects are present, the study is stopped and the Maximum Tolerated Dose (MTD) is declared.

Group 1 and group 2 were used to identify the MTD of CART-PSMA-TGF β RDN cells. MTD was defined as the highest dose at which 0/3 or 1/6DLT appeared.

Group 3 subjects (N-3 or 6) received a single infusion of lentivirus transduced CART-PSMA-TGF β RDN cells at day 0 with MTD, followed by a single dose of 1.0 g/m 4 days (day-3 ± 1) before CAR T cells²Cyclophosphamide of (1). If 0 DLT/3 subjects are present, the study enrolls an additional 3 patients to confirm tolerance. If 1 DLT/3 subject is present, the study enrolls an additional 3 subjects at this dose level. If two of the first three subjects experienced DLT, the dose of lymphoablative chemotherapy administered up to 4 days (day-3. + -. 1) before CAR T cells was reduced to 500mg/m ²。

Subjects were enrolled continuously. Staggered infusions allowed assessment of group progression, expansion or dose-reduced DLT. The infusions of the first two subjects in each group were staggered by 28 days; the second subject was not infused until 28 days after the infusion of the first subject. Subjects 2 and 3 in each group were infused and tracked in parallel, but only started when subject 1 in the group completed the day 28 visit without a DLT.

DLT is defined as any new grade 3 or higher adverse event occurring within 28 days of T cell infusion that may be at least relevant to T cell protocols. If 1 DLT occurs in the first 3 subjects treated at the dose level, the study enrolled 3 additional subjects at that dose level. If 2 DLT/3 subjects are present, the study is stopped and the maximum tolerated dose is declared, except for group 1, where a 10-fold dose reduction occurs. If at this dose level 0 DLT/3 subjects or 1 DLT/6 subjects, then the study progressed to the next group. For group 3, if two of the first three subjects experienced DLT, the dose of lymphodepleting chemotherapy administered up to 4 days (day-3 ± 1) before CAR T cells was reduced to 500mg/m for the other three patients ². Otherwise, if 0-1 DLT/3 subjects appeared in group 3, the study was enrolledAn additional 3 patients to confirm tolerability.

Subjects were evaluated for safety and study. For safety assessment, subjects returned for study follow-up on days 1, 3, 7, 10, 14, 21 and 28. On day 28 (± 5), disease staging was performed with CT of chest/abdomen/pelvis, bone scan and serum PSA. The reason for this early imaging assessment at day 28 was to assess systemic inflammatory effects and monitor the disease state when CART-PSMA-TGF β RDN cells are expected to home (homing). Repeat disease assessments (including imaging) were performed at month 3 and month 6 and thereafter as a standard of care. If a subject has relevant imaging data (CT abd/pelvis, MRI abd/pelvis, bone scan) as part of their standard of care within 4 weeks of month 3 and/or month 6, it is not repeated at month 3 and/or month 6.

Adverse event reporting was initiated at consent and continued until the subjects ended the study. During the study, subjects continually reassess for evidence of acute and cumulative toxicity. Following interruption from the primary follow-up phase, subjects entered a follow-up period of up to 5 years from their CART-PSMA-TGF β RDN infusion. During long-term follow-up, subjects were monitored for delayed adverse events that may be associated with administration of CART-PSMA-TGF β RDN cells.

Peripheral blood samples were obtained at defined time points to monitor measurements of safety and efficacy. Additional blood and tissue samples (e.g., fluid, tissue biopsy) obtained for the clinical indication may also be sent for study analysis. At any time a tissue or body fluid is obtained (e.g., drainage of pleural or ascites), the fluid sample, which is otherwise discarded, is used for research purposes. These studies include, but are not limited to, CART-PSMA-TGF β RDN cell persistence studies by Q-PCR and inflammatory marker evaluation using Luminex-based cytokine and chemokine panels.

In the case of unexpected AEs, additional blood and tissue were collected for study analysis, focusing on evaluating potential causal relationships of unexpected events of infused CART-PSMA-TGF β RDN cells. No more than 3 tablespoons of blood were collected twice a week for the additional samples used for the study, and the tissue sample collection procedure was performed once a month.

Inclusion criteria were:

1. metastatic castration resistant prostate cancer

2. As confirmed by immunohistochemical analysis of biopsied tissue, > 10% of PSMA expressing tumor cells were present.

3. Radiographic evidence of bone metastatic disease and/or measurable non-bone metastatic disease (lymph nodes or internal organs)

4. Patient >18 years old

The expression state of ECOG is 0-1

6. Adequate organ function is defined as follows:

a. serum creatinine is less than or equal to 1.5mg/dl or creatinine clearance is more than or equal to 60cc/min

b. Serum total bilirubin <1.5x ULN

c. Serum ALT/AST <2x ULN

7. Appropriate hematological characteristics were found within 4 weeks after study enrollment, defined as follows:

a.Hgb>10g/dl

b.PLT>100k/ul

c.ANC>1.5k/ul

note that: subject independent transfusion

8. Evidence for progressive castration resistant prostate cancer is defined as follows:

a. castration levels of testosterone with or without androgen deprivation therapy (<50ng/ml)

b. Within 12 weeks prior to study enrollment, there was evidence of one of the following progressive diseases:

i. soft tissue progression according to RECIST 1.1 criteria

Bone disease progression with 2 or more new lesions in bone scan (according to PCWG2 standard)

An increase in serum PSA of at least 25% and an absolute nadir increase of 2ng/ml or more (according to PCWG2 standard)

9. Metastatic castration resistant prostate cancer has previously been treated with at least one standard 17 alpha lyase inhibitor or a second generation anti-androgen therapy

10. Providing written informed consent

11. Subjects with reproductive potential must agree to use an acceptable method of contraception.

Exclusion criteria:

1. previous treatments used immune-based therapies for prostate cancer, including cancer vaccine therapies (such as SipuleucelT, PROSTVAC), immune checkpoint inhibitors, radium-223, and immunoconjugate therapies

2. Active non-curative non-prostate primary malignancy history in the last 5 years

3. Subjects in need of long-term treatment with systemic corticosteroids

4. Subjects who have received >3 prior therapies for castration-resistant prostate cancer (not including luteinizing hormone-releasing hormone agonists or antagonists, or a generation of anti-androgen therapy). This includes subjects receiving a taxane in a non-castration resistant environment.

5. Subjects classified as class III/IV cardiovascular disease according to the New York Heart Association (see appendix 2)

6. Subjects with symptomatic spinal metastases affecting spinal cord function (determined from clinical history, physical examination, or MRI imaging)

7. History of active autoimmune diseases in need of immunosuppressive therapy

8. Persistent or active infection.

9. The study product excipients (human serum albumin, DMSO and dextran 40) had a history of allergy or hypersensitivity

10. Active hepatitis B, hepatitis C or HIV infection.

Example 10: phase 1 clinical safety data

By 2018, 7 months, 25 days, a total of 6 subjects were infused and two subjects were still under study. 3 subjects were infused in group 1 and 3 subjects were infused in group 2. Thus, group 2 is full. All three subjects infused in group 2 experienced Cytokine Release Syndrome (CRS) compared to group 1 (extract): two subjects had grade 3 CRS and one subject had grade 1 CRS, all occurring within 12 hours of CAR T cell infusion. These toxicities were managed and resolved according to protocol/institutional guidelines. Thus, group 2 is completed without a DLT.

Study site initiated visits were held on wednesday 22/month 2.2017, and the study was initiated on 8/month 3.2017. By 7 months and 25 days 2018, 8 subjects were agreed to in the clinical site. Of the 8 subjects who agreed, 1 failed the screen, 1 was withdrawn prior to treatment, and 6 subjects were infused.

Table 3 shows a demographic summary of the screened subjects (N ═ 8). .

Table 3: demographics of screened subjects

Not applicable to N/A

Death occurred during long-term follow-up. Therefore, this event does not meet the requirements of PDAE and is determined to be IP independent.

Table 4 shows a summary of the current protocol status of the infused subjects (N-6).

Table 4: current protocol status of infused subjects

Table 5 shows a summary of the deviations or exceptions (N ═ 6) of the infused subjects.

Table 5: deviation or exception of infused subjects

Table 6 shows a summary of infusion dates and doses in the infused subjects (N ═ 6).

Table 6: test for infusionPSMA-TGF beta RDN infusion date and dosage in a subject

Table 7 is a summary of disease responses of infused subjects (N ═ 6).

Table 7: disease response in infused subjects

NE is not evaluable

Progressive disease of PD

Stable disease with SD ═

Pending as the subject has not reached the time point

Not evaluated-no evaluation was made at this 1 time point

N/a ═ not applicable/subjects terminated primary tracking before this time point 1.

Table 8 is a summary of serum PSA levels for infused subjects (N ═ 6).

Table 8: serum PSA level in infused patients (data provided in ng/mL)

Not applicable to N/A

Subject entered LTFU at 2017/11/1; month 2 PSA plotted at 2017/11/2

No unplanned data on this subject

The subject has not reached this point in time

Table 9 is a summary showing PSMA-TGF β RDN cells labeled in peripheral blood by qPCR for infused subjects (N ═ 6).

Table 9: subjects infused were scored in peripheral blood by qPCRNote PSMA-TGF beta RDN cells (in copies/micro) Gram genomic DNA as a unit to provide data

ND is not detected

N/a not applicable-subject terminates primary tracking before this visit

The subject still has not reached this point in time

Study samples were not collected for analysis

No results-samples have not been tested

Table 10 is a summary showing PSMA-TGF β RDN cells labeled by qPCR for infused subjects in other tissues (N ═ 6).

Table 10: PSMA-TGF β RDN cells (in copy `) labeled in other tissues by qPCR on infused subjects Microgram genomic DNA as a Unit supply data

FFPE (FFPE) -formalin fixation and paraffin embedding

BMBMX bone marrow biopsy

BX ═ biopsy

ND is not detected

Table 11 is a summary showing the percentage of enrolled subject PSMA-positive tumor cells as determined by immunohistochemistry (N-7).

Table 11: percentage of PSMA-positive tumor cells in enrolled subjects

NA is unspecified

ND is not detected

Example 11: PSMA-directed/TGF-beta insensitive CAR-T cells in metastatic castration-resistant prostate cancer Phase 1 clinical trial

Background:

adoptive immunotherapy using CAR-T cells has the transforming potential to treat cancer. The major challenge in the success of these treatments in prostate cancer is the immunosuppressive microenvironment, including the high levels of TGF β encountered by redirected T cells following tumor infiltration. Importantly, the use of dominant negative TGF β receptor (TGF β Rdn) can suppress these immunosuppressive functions of TGF β in T cells, thereby enhancing anti-tumor immunity. In an in vivo disseminated tumor model, co-expression of TGF β Rdn on PSMA-directed CAR-T cells resulted in increased T cell proliferation, enhanced cytokine secretion, long-term persistence, and greater induction of tumor eradication. The mechanism of adoptive tumor resistance is not clear.

FIG. 15 shows the efficacy of CART-PSMA-TGF β Rdn cells in a tumor model spread in vivo. Figure 15A is a graph showing that CART-PSMA-TGF β Rdn cells exhibit enhanced antigen-specific proliferation relative to CART-PSMA in 42 days of co-culture and repeated stimulation with PSMA-expressing tumor cells. Figure 15B is a graph showing that CART-PSMA-TGF β Rdn cells in vivo showed significantly increased tumor reduction compared to CART-PSMA, as measured by weekly BLI imaging to assess tumor burden. Fig. 15C is a photograph showing the location and systemic burden of weekly BLI assessed tumors. Abbreviations used in fig. 15: pbbz ═ CAR-T PSMA; dnTGFBR2-T2A-Pbbz ═ CART-PSMA-TGF β Rdn; 19bbz ═ anti-CD 19 CAR.

Research and design:

summary of the study: a first phase 1 clinical trial in humans was conducted to evaluate the safety and primary efficacy of lentivirus transduced PSMA-directed/TGF β insensitive CAR-T cells (CART-PSMA-TGF β Rdn) in treating men with refractory metastatic Castration Resistant Prostate Cancer (CRPC) (NCT 03089203). In the preliminary dose escalation group, patients received a single dose of 1-3x10⁷/m²(group ofOi 1) or 1-3x10⁸/m²(group 2) CART-PSMA-TGF β Rdn cells without the lymphodepleting chemotherapy in the 3+3 null design. In group 3, the patient was on cyclophosphamide 300mg/m ²And fludarabine 30mg/m²CART-PSMA-TGF β Rdn cells received a Maximum Tolerated Dose (MTD) 3 days after undergoing lymphoablative chemotherapy. All treated patients received metastatic tumor biopsies at baseline and +10 days post CAR-T cell infusion.

Key specification: metastatic CRPC previously treated with at least one second generation androgen signaling inhibitor (abiraterone or enzalutamide); when the tissue biopsy of the metastatic tissue is inspected by IHC, more than or equal to 10 percent of tumor cells express PSMA; radiographic evidence of metastatic disease (bone or lymph nodes/internal organs); metastatic CRPC treatment is less than or equal to 4 lines.

The research scheme is as follows: figure 16 shows the study protocol used in this clinical trial.

And (3) correlation analysis: quantitative PCR of CART-PSMA-TGF β rdnddna was performed at successive time points to assess CAR-T amplification and persistence in peripheral blood and trafficking to target tissues. The biological activity of CART-PSMA-TGF β Rdn cells in peripheral blood was assessed by Luminex analysis of immune and inflammatory factors. Circulating tumor material was collected at successive time points and correlated with clinical response.

State of the art and preliminary findings: 6 patients received a CART-PSMA-TGF β Rdn cell infusion at the indicated dose level (group 1, N ═ 3; group 2, N ═ 3). All CART-PSMA-TGF β Rdn infusion products met the target transduction efficiency. No dose limiting toxicity was observed in the initial dose escalation.

Assessment of CAR-T cell kinetics by qPCR of CART-PSMA-TGF β rdnddna confirmed the expansion of peripheral blood T cells (fig. 17), as well as tumor tissue trafficking in post-treatment tumor biopsies (table 17).

Table 17: CART-PSMA-TGF β RDN cell trafficking: qPCR detection in tissue biopsy samples of infused subjects

Copy/ug gDNA

FFPE (FFPE) -formalin fixation and paraffin embedding

ND is not detected

In group 2, two patients developed the expected grade 3 Cytokine Release Syndrome (CRS), a key marker of the biological activity of CAR-T therapy, and one patient developed grade 3 CAR-T neurotoxicity requiring corticosteroids.

Significant increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFN γ) and ferritin were associated with all grade 3 CRS events (subject 32816-06: FIG. 18A; and subject 32816-07: FIG. 18B). All CRS events were rapidly resolved with tositumumab (anti-5 IL6R) rescue.

Group 3 enrollment (MTD with lymphodepleting chemotherapy) began in 2018 at 9 months.

Example 12: group 1 and 2 observations and case study

Figure 19 shows a graph of Prostate Specific Antigen (PSA) responses in group 1 and group 2 patients.

Subject 32816-07: age 74 has metastatic castration resistant prostate cancer (mCRPC; preliminary diagnosis: 5 months 2014). Fever to 103F (no lymphocyte clearance) was observed several hours after PSMA-TGF β RDN CART infusion. Hypotension was observed approximately 6 hours after PSMA-TGF β RDN CART infusion, with a nadir of 83/44 mmHg. The following day after infusion of PSMA-TGF β RDN CART, hypotension was managed with crystalloid infusion (no pharmacological management required during ICU hospitalization) and tosubuzumab.

Cytokine syndrome (CRS) was observed in patient 32816-07 following PSMA-TGF β RDN CART infusion (fig. 20A). In FIG. 20A, the left y-axis represents PSMA-TGF β RDN CART levels in peripheral blood in copies/ug genomic DNA (32816-07), and the right y-axis represents IL-6 levels in pg/ml (IL 6). CRS was accompanied by a transient decrease in PSA (fig. 20B). In FIG. 20B, the left y-axis represents serum levels of C-reactive protein (CRP) in mg/L and the right y-axis represents serum levels of ferritin in ng/L.

PSMA positive CTC observations in groups 1 and 2: table 18 shows a summary of the number of PSMA positive Circulating Tumor Cells (CTCs) detected in each subject at different time points, the data of which are plotted in fig. 21.

Table 18: PSMA-positive CTCs in groups 1 and 2

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the following claims be interpreted to include all such embodiments and equivalent variations.

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180 185 190

Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe

210 215 220

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

225 230 235

<210> 14

<211> 260

<212> PRT

<213> mouse

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Asp Ile Val Met Thr Gln Ser His Lys

20 25 30

Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala

35 40 45

Ser Gln Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly

65 70 75 80

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln

100 105 110

Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp

115 120 125

Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly

130 135 140

Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

145 150 155 160

Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu

165 170 175

Tyr Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp

180 185 190

Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys

195 200 205

Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala

210 215 220

Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225 230 235 240

Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

245 250 255

Thr Val Ser Ser

260

<210> 15

<211> 780

<212> DNA

<213> mouse

<400> 15

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca cgccgccaga 60

cctggatctg acattgtgat gacccagtct cacaaattca tgtccacatc agtaggagac 120

agggtcagca tcatctgtaa ggccagtcaa gatgtgggta ctgctgtaga ctggtatcaa 180

cagaaaccag gacaatctcc taaactactg atttattggg catccactcg gcacactgga 240

gtccctgatc gcttcacagg cagtggatct gggacagact tcactctcac cattactaac 300

gttcagtctg aagacttggc agattatttc tgtcagcaat ataacagcta tcctctcacg 360

ttcggtgctg ggaccatgct ggacctgaaa ggaggcggag gatctggcgg cggaggaagt 420

tctggcggag gcagcgaggt gcagctgcag cagagcggac ccgagctcgt gaagcctgga 480

acaagcgtgc ggatcagctg caagaccagc ggctacacct tcaccgagta caccatccac 540

tgggtcaagc agtcccacgg caagagcctg gagtggatcg gcaatatcaa ccccaacaac 600

ggcggcacca cctacaacca gaagttcgag gacaaggcca ccctgaccgt ggacaagagc 660

agcagcaccg cctacatgga actgcggagc ctgaccagcg aggacagcgc cgtgtactat 720

tgtgccgccg gttggaactt cgactactgg ggccagggca caaccctgac agtgtctagc 780

<210> 16

<211> 107

<212> PRT

<213> mouse

<400> 16

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys

100 105

<210> 17

<211> 321

<212> DNA

<213> mouse

<400> 17

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcatctgta aggccagtca agatgtgggt actgctgtag actggtatca acagaaacca 120

ggacaatctc ctaaactact gatttattgg gcatccactc ggcacactgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagac ttcactctca ccattactaa cgttcagtct 240

gaagacttgg cagattattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300

gggaccatgc tggacctgaa a 321

<210> 18

<211> 11

<212> PRT

<213> mouse

<400> 18

Lys Ala Ser Gln Asp Val Gly Thr Ala Val Asp

1 5 10

<210> 19

<211> 7

<212> PRT

<213> mouse

<400> 19

Trp Ala Ser Thr Arg His Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> mouse

<400> 20

Gln Gln Tyr Asn Ser Tyr Pro Leu Thr

1 5

<210> 21

<211> 115

<212> PRT

<213> mouse

<400> 21

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 22

<211> 345

<212> DNA

<213> mouse

<400> 22

gaggtgcagc tgcagcagag cggacccgag ctcgtgaagc ctggaacaag cgtgcggatc 60

agctgcaaga ccagcggcta caccttcacc gagtacacca tccactgggt caagcagtcc 120

cacggcaaga gcctggagtg gatcggcaat atcaacccca acaacggcgg caccacctac 180

aaccagaagt tcgaggacaa ggccaccctg accgtggaca agagcagcag caccgcctac 240

atggaactgc ggagcctgac cagcgaggac agcgccgtgt actattgtgc cgccggttgg 300

aacttcgact actggggcca gggcacaacc ctgacagtgt ctagc 345

<210> 23

<211> 10

<212> PRT

<213> mouse

<400> 23

Gly Tyr Thr Phe Thr Glu Tyr Thr Ile His

1 5 10

<210> 24

<211> 17

<212> PRT

<213> mouse

<400> 24

Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu

1 5 10 15

Asp

<210> 25

<211> 6

<212> PRT

<213> mouse

<400> 25

Gly Trp Asn Phe Asp Tyr

1 5

<210> 26

<211> 267

<212> PRT

<213> Intelligent people

<400> 26

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ala Val Pro Trp Gly Ser Arg Tyr Tyr

115 120 125

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

165 170 175

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile

195 200 205

Phe Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

225 230 235 240

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

245 250 255

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

260 265

<210> 27

<211> 801

<212> DNA

<213> Intelligent people

<400> 27

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 120

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 180

gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa caataaatac 240

tacgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 300

tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc 360

gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg 420

gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 480

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 540

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaatca 600

gggaaagctc ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca 660

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720

gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga 780

gggaccaagg tggagatcaa a 801

<210> 28

<211> 125

<212> PRT

<213> Intelligent people

<400> 28

Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly

1 5 10 15

Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser

20 25 30

Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ala Val Pro Trp Gly Ser Arg Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 375

<212> DNA

<213> Intelligent people

<400> 29

ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 60

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 120

gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa caataaatac 180

tacgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 240

tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc 300

gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg 360

gtcaccgtct cctca 375

<210> 30

<211> 5

<212> PRT

<213> Intelligent people

<400> 30

Ser Tyr Ala Met His

1 5

<210> 31

<211> 17

<212> PRT

<213> Intelligent people

<400> 31

Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 32

<211> 15

<212> PRT

<213> Intelligent people

<400> 32

Ala Val Pro Trp Gly Ser Arg Tyr Tyr Tyr Tyr Gly Met Asp Val

1 5 10 15

<210> 33

<211> 107

<212> PRT

<213> Intelligent people

<400> 33

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 34

<211> 321

<212> DNA

<213> Intelligent people

<400> 34

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaatca 120

gggaaagctc ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 35

<211> 11

<212> PRT

<213> Intelligent people

<400> 35

Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala

1 5 10

<210> 36

<211> 7

<212> PRT

<213> Intelligent people

<400> 36

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 37

<211> 9

<212> PRT

<213> Intelligent people

<400> 37

Gln Gln Phe Asn Ser Tyr Pro Leu Thr

1 5

<210> 38

<211> 262

<212> PRT

<213> Intelligent people

<400> 38

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Ser Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys

260

<210> 39

<211> 786

<212> DNA

<213> Intelligent people

<400> 39

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 360

actggtttcc tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcaacaga aaccagggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggctat 660

ggatctggga cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaa 786

<210> 40

<211> 120

<212> PRT

<213> Intelligent people

<400> 40

Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

1 5 10 15

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

20 25 30

Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

50 55 60

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

65 70 75 80

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Ser Asp Leu Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 41

<211> 360

<212> DNA

<213> Intelligent people

<400> 41

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 60

atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag 120

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 180

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 240

tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 300

actggtttcc tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca 360

<210> 42

<211> 5

<212> PRT

<213> Intelligent people

<400> 42

Ser Asn Trp Ile Gly

1 5

<210> 43

<211> 17

<212> PRT

<213> Intelligent people

<400> 43

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 44

<211> 10

<212> PRT

<213> Intelligent people

<400> 44

Gln Thr Gly Phe Leu Trp Ser Ser Asp Leu

1 5 10

<210> 45

<211> 107

<212> PRT

<213> Intelligent people

<400> 45

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Tyr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 321

<212> DNA

<213> Intelligent people

<400> 46

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggacattagc agtgctttag cctggtatca acagaaacca 120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180

aggttcagcg gctatggatc tgggacagat ttcactctca ccatcaacag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 47

<211> 11

<212> PRT

<213> Intelligent people

<400> 47

Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu

1 5 10

<210> 48

<211> 8

<212> PRT

<213> Intelligent people

<400> 48

Tyr Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 49

<211> 10

<212> PRT

<213> Intelligent people

<400> 49

Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr

1 5 10

<210> 50

<211> 264

<212> PRT

<213> Intelligent people

<400> 50

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Ile Lys

260

<210> 51

<211> 792

<212> DNA

<213> Intelligent people

<400> 51

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaatca aa 792

<210> 52

<211> 120

<212> PRT

<213> Intelligent people

<400> 52

Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly

1 5 10 15

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser

20 25 30

Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

50 55 60

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

65 70 75 80

Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp Leu Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 53

<211> 360

<212> DNA

<213> Intelligent people

<400> 53

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 60

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 120

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 180

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 240

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 300

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 360

<210> 54

<211> 5

<212> PRT

<213> Intelligent people

<400> 54

Ser Asn Trp Ile Gly

1 5

<210> 55

<211> 17

<212> PRT

<213> Intelligent people

<400> 55

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 56

<211> 10

<212> PRT

<213> Intelligent people

<400> 56

Gln Thr Gly Phe Leu Trp Ser Phe Asp Leu

1 5 10

<210> 57

<211> 109

<212> PRT

<213> Intelligent people

<400> 57

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys

100 105

<210> 58

<211> 327

<212> DNA

<213> Intelligent people

<400> 58

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca ggacattagc agtgctttag cctggtatca gcagaaaccg 120

gggaaagctc ctaagctcct gatctatgat gcctccagtt tggaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttatta ctgtcaacag tttaatagtt acccgctcac tttcggcgga 300

gggaccaagg tggagatcaa aatcaaa 327

<210> 59

<211> 11

<212> PRT

<213> Intelligent people

<400> 59

Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala

1 5 10

<210> 60

<211> 7

<212> PRT

<213> Intelligent people

<400> 60

Asp Ala Ser Ser Leu Glu Ser

1 5

<210> 61

<211> 9

<212> PRT

<213> Intelligent people

<400> 61

Gln Gln Phe Asn Ser Tyr Pro Leu Thr

1 5

<210> 62

<211> 265

<212> PRT

<213> Intelligent people

<400> 62

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ser Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val

145 150 155 160

Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala

165 170 175

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

195 200 205

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

225 230 235 240

Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Phe Thr Phe

245 250 255

Gly Pro Gly Thr Lys Val Asp Ile Lys

260 265

<210> 63

<211> 795

<212> DNA

<213> Intelligent people

<400> 63

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 360

gggtatacca gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc 420

tcctcaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaaattgtg 480

ttgacacagt ctccagccac cctgtctttg tctccagggg aaagagccac cctctcctgc 540

agggccagtc agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct 600

cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc caggttcagt 660

ggcagtgggt ctgggacaga cttcactctc accatcagca gcctagagcc tgaagatttt 720

gcagtttatt actgtcagca gcgtagcaac tggcccctat tcactttcgg ccctgggacc 780

aaagtggata tcaaa 795

<210> 64

<211> 122

<212> PRT

<213> Intelligent people

<400> 64

Pro Glu Val Gln Leu Val Gln Ser Gly Ser Glu Val Lys Lys Pro Gly

1 5 10 15

Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn

20 25 30

Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser

50 55 60

Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala

65 70 75 80

Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr

85 90 95

Cys Ala Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 366

<212> DNA

<213> Intelligent people

<400> 65

ccggaggtgc agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag 60

atctcctgta agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag 120

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 180

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 240

tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 300

gggtatacca gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc 360

tcctca 366

<210> 66

<211> 4

<212> PRT

<213> Intelligent people

<400> 66

Thr Asn Tyr Trp

1

<210> 67

<211> 18

<212> PRT

<213> Intelligent people

<400> 67

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

1 5 10 15

Gln Gly

<210> 68

<211> 10

<212> PRT

<213> Intelligent people

<400> 68

Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser

1 5 10

<210> 69

<211> 108

<212> PRT

<213> Intelligent people

<400> 69

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 70

<211> 324

<212> DNA

<213> Intelligent people

<400> 70

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcccctatt cactttcggc 300

cctgggacca aagtggatat caaa 324

<210> 71

<211> 11

<212> PRT

<213> Intelligent people

<400> 71

Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu

1 5 10

<210> 72

<211> 8

<212> PRT

<213> Intelligent people

<400> 72

Tyr Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 73

<211> 11

<212> PRT

<213> Intelligent people

<400> 73

Cys Gln Gln Arg Ser Asn Trp Pro Leu Phe Thr

1 5 10

<210> 74

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 74

Asp Lys Thr His Thr

1 5

<210> 75

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 75

Cys Pro Pro Cys

1

<210> 76

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 76

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

1 5 10 15

<210> 77

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 77

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr

1 5 10

<210> 78

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 78

Lys Ser Cys Asp Lys Thr His Thr Cys Pro

1 5 10

<210> 79

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 79

Lys Cys Cys Val Asp Cys Pro

1 5

<210> 80

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 80

Lys Tyr Gly Pro Pro Cys Pro

1 5

<210> 81

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 81

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 82

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 82

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> 83

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 83

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys

1 5 10 15

Pro

<210> 84

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 84

Ser Pro Asn Met Val Pro His Ala His His Ala Gln

1 5 10

<210> 85

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 85

Glu Pro Lys Ser Cys Asp Lys Thr Tyr Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 86

<211> 45

<212> PRT

<213> Artificial sequence

<220>

<223> CD8 hinge

<400> 86

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 87

<211> 135

<212> DNA

<213> Artificial sequence

<220>

<223> CD8 hinge

<400> 87

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 88

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> transmembrane domain of CD8

<400> 88

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 89

<211> 72

<212> DNA

<213> Artificial sequence

<220>

<223> transmembrane domain of CD8

<400> 89

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 90

<211> 69

<212> PRT

<213> Artificial sequence

<220>

<223> CD8 hinge and CD8 transmembrane domain

<400> 90

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 91

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223> CD8 hinge and CD8 transmembrane domain

<400> 91

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 92

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB

<400> 92

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 93

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> 4-1BB

<400> 93

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagacgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 94

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> 4-1BB

<400> 94

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 95

<211> 35

<212> PRT

<213> Artificial sequence

<220>

<223> ICOS(YMNM)

<400> 95

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 96

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> ICOS(YMNM)

<400> 96

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gaacatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105

<210> 97

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 zeta chain

<400> 97

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 98

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> CD3 zeta chain

<400> 98

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 99

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> CD3 zeta chain

<400> 99

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgacg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaac 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga cggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 100

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 zeta chain

<400> 100

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 101

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> CD3 zeta chain

<400> 101

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 102

<211> 154

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB Domain and CD3 zeta chain

<400> 102

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

35 40 45

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

50 55 60

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

65 70 75 80

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

85 90 95

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

100 105 110

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

115 120 125

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

130 135 140

Ala Leu His Met Gln Ala Leu Pro Pro Arg

145 150

<210> 103

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> 4-1BB Domain and CD3 zeta chain

<400> 103

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagacgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 180

cagctctata acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga 240

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 300

tacaacgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 360

gagcgccgga ggggcaaggg gcacgacggc ctttaccagg gtctcagtac agccaccaag 420

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 462

<210> 104

<211> 462

<212> DNA

<213> Artificial sequence

<220>

<223> 4-1BB Domain and CD3 zeta chain

<400> 104

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 180

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 240

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 300

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 360

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 420

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 462

<210> 105

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<223> J591 murine PSMA-CAR

<400> 105

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gly Ser Asp Ile Val Met Thr Gln Ser His Lys

20 25 30

Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Ile Cys Lys Ala

35 40 45

Ser Gln Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr Gly

65 70 75 80

Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln

100 105 110

Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp

115 120 125

Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly

130 135 140

Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

145 150 155 160

Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu

165 170 175

Tyr Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp

180 185 190

Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys

195 200 205

Phe Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala

210 215 220

Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225 230 235 240

Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

245 250 255

Thr Val Ser Ser Ala Ser Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

325 330 335

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

340 345 350

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

355 360 365

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

385 390 395 400

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

405 410 415

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

420 425 430

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

435 440 445

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

450 455 460

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

465 470 475 480

Met Gln Ala Leu Pro Pro Arg

485

<210> 106

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<223> J591 murine PSMA-CAR

<400> 106

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca cgccgccaga 60

cctggatctg acattgtgat gacccagtct cacaaattca tgtccacatc agtaggagac 120

agggtcagca tcatctgtaa ggccagtcaa gatgtgggta ctgctgtaga ctggtatcaa 180

cagaaaccag gacaatctcc taaactactg atttattggg catccactcg gcacactgga 240

gtccctgatc gcttcacagg cagtggatct gggacagact tcactctcac cattactaac 300

gttcagtctg aagacttggc agattatttc tgtcagcaat ataacagcta tcctctcacg 360

ttcggtgctg ggaccatgct ggacctgaaa ggaggcggag gatctggcgg cggaggaagt 420

tctggcggag gcagcgaggt gcagctgcag cagagcggac ccgagctcgt gaagcctgga 480

acaagcgtgc ggatcagctg caagaccagc ggctacacct tcaccgagta caccatccac 540

tgggtcaagc agtcccacgg caagagcctg gagtggatcg gcaatatcaa ccccaacaac 600

ggcggcacca cctacaacca gaagttcgag gacaaggcca ccctgaccgt ggacaagagc 660

agcagcaccg cctacatgga actgcggagc ctgaccagcg aggacagcgc cgtgtactat 720

tgtgccgccg gttggaactt cgactactgg ggccagggca caaccctgac agtgtctagc 780

gctagctccg gaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagacggc 1080

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgacgttttg gacaagagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaacgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgacggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg c 1461

<210> 107

<211> 490

<212> PRT

<213> Artificial sequence

<220>

<223> 1C3 human PSMA-CAR

<400> 107

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Asn Asn Lys Tyr

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ala Val Pro Trp Gly Ser Arg Tyr Tyr

115 120 125

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

165 170 175

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

180 185 190

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu Leu Ile

195 200 205

Phe Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

225 230 235 240

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

245 250 255

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

340 345 350

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

355 360 365

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

370 375 380

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

385 390 395 400

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

405 410 415

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

420 425 430

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

435 440 445

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

450 455 460

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 108

<211> 1470

<212> DNA

<213> Artificial sequence

<220>

<223> 1C3 human PSMA-CAR

<400> 108

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 120

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 180

gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa caataaatac 240

tacgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 300

tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc 360

gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg 420

gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 480

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 540

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaatca 600

gggaaagctc ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca 660

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720

gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga 780

gggaccaagg tggagatcaa aaccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagacggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gacgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caacgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgacggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc 1470

<210> 109

<211> 485

<212> PRT

<213> Artificial sequence

<220>

<223> 2A10 human PSMA-CAR

<400> 109

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Ser Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Tyr Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

260 265 270

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

275 280 285

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

290 295 300

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

305 310 315 320

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

325 330 335

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

340 345 350

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

355 360 365

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

370 375 380

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

385 390 395 400

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

405 410 415

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

420 425 430

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

435 440 445

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

450 455 460

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

465 470 475 480

Ala Leu Pro Pro Arg

485

<210> 110

<211> 1455

<212> DNA

<213> Artificial sequence

<220>

<223> 2A10 human PSMA-CAR

<400> 110

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 360

actggtttcc tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcaacaga aaccagggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggctat 660

ggatctggga cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1020

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga cggctgtagc 1080

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1140

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1200

cgaagagagg agtacgacgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaacg aactgcagaa agataagatg 1320

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgac 1380

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440

gccctgcccc ctcgc 1455

<210> 111

<211> 487

<212> PRT

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR

<400> 111

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

325 330 335

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

340 345 350

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

355 360 365

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

385 390 395 400

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

405 410 415

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

420 425 430

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

435 440 445

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

450 455 460

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

465 470 475 480

Met Gln Ala Leu Pro Pro Arg

485

<210> 112

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR

<400> 112

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagacggc 1080

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgacgttttg gacaagagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaacgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgacggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg c 1461

<210> 113

<211> 488

<212> PRT

<213> Artificial sequence

<220>

<223> 2C6 human PSMA-CAR

<400> 113

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ser Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Asn Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Ser Pro Gly Tyr Thr Ser Ser Trp Thr Ser

115 120 125

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val

145 150 155 160

Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala

165 170 175

Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala

195 200 205

Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

225 230 235 240

Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu Phe Thr Phe

245 250 255

Gly Pro Gly Thr Lys Val Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 114

<211> 1464

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6 human PSMA-CAR

<400> 114

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 360

gggtatacca gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc 420

tcctcaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaaattgtg 480

ttgacacagt ctccagccac cctgtctttg tctccagggg aaagagccac cctctcctgc 540

agggccagtc agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct 600

cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc caggttcagt 660

ggcagtgggt ctgggacaga cttcactctc accatcagca gcctagagcc tgaagatttt 720

gcagtttatt actgtcagca gcgtagcaac tggcccctat tcactttcgg ccctgggacc 780

aaagtggata tcaaaaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagac 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgacgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaacga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgacg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgc 1464

<210> 115

<211> 201

<212> PRT

<213> Artificial sequence

<220>

<223> TGFBRII-DN

<400> 115

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Cys Tyr Arg Val Asn

180 185 190

Arg Gln Gln Lys Leu Ser Ser Ser Gly

195 200

<210> 116

<211> 603

<212> DNA

<213> Artificial sequence

<220>

<223> TGFBRII-DN

<400> 116

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

gga 603

<210> 117

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28 transducible receptor

<400> 117

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Phe Trp Val Leu Val Val

165 170 175

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

180 185 190

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

195 200 205

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

210 215 220

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

225 230 235

<210> 118

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28 transducible receptor

<400> 118

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctcc 714

<210> 119

<211> 232

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-PTM-CD28 conversion receptor

<400> 119

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg

180 185 190

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

195 200 205

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

210 215 220

Arg Asp Phe Ala Ala Tyr Arg Ser

225 230

<210> 120

<211> 696

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-PTM-CD28 conversion receptor

<400> 120

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctcc 696

<210> 121

<211> 232

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28 transducible receptor

<400> 121

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg

180 185 190

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

195 200 205

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

210 215 220

Arg Asp Phe Ala Ala Tyr Arg Ser

225 230

<210> 122

<211> 693

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28 transducible receptor

<400> 122

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgc 693

<210> 123

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> TGFBR-IL12RB1 receptor

<400> 123

Met Glu Ala Ala Val Ala Ala Pro Arg Pro Arg Leu Leu Leu Leu Val

1 5 10 15

Leu Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Leu Pro Gly Ala Thr

20 25 30

Ala Leu Gln Cys Phe Cys His Leu Cys Thr Lys Asp Asn Phe Thr Cys

35 40 45

Val Thr Asp Gly Leu Cys Phe Val Ser Val Thr Glu Thr Thr Asp Lys

50 55 60

Val Ile His Asn Ser Met Cys Ile Ala Glu Ile Asp Leu Ile Pro Arg

65 70 75 80

Asp Arg Pro Phe Val Cys Ala Pro Ser Ser Lys Thr Gly Ser Val Thr

85 90 95

Thr Thr Tyr Cys Cys Asn Gln Asp His Cys Asn Lys Ile Glu Leu Pro

100 105 110

Thr Thr Val Lys Ser Ser Pro Gly Leu Gly Pro Val Glu Leu Ala Ala

115 120 125

Val Ile Ala Gly Pro Val Cys Phe Val Cys Ile Ser Leu Met Leu Met

130 135 140

Val Tyr Ile Arg Ala Ala Arg His Leu Cys Pro Pro Leu Pro Thr Pro

145 150 155 160

Cys Ala Ser Ser Ala Ile Glu Phe Pro Gly Gly Lys Glu Thr Trp Gln

165 170 175

Trp Ile Asn Pro Val Asp Phe Gln Glu Glu Ala Ser Leu Gln Glu Ala

180 185 190

Leu Val Val Glu Met Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro Leu

195 200 205

Glu Lys Thr Glu Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp Thr

210 215 220

Glu Leu Ser Leu Glu Asp Gly Asp Arg Cys Lys Ala Lys Met

225 230 235

<210> 124

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<223> TGFBR-IL12RB1 receptor

<400> 124

atggaggcgg cggtcgctgc tccgcgtccc cggctgctcc tcctcgtgct ggcggcggcg 60

gcggcggcgg cggcggcgct gctcccgggg gcgacggcgt tacagtgttt ctgccacctc 120

tgtacaaaag acaattttac ttgtgtgaca gatgggctct gctttgtctc tgtcacagag 180

accacagaca aagttataca caacagcatg tgtatagctg aaattgactt aattcctcga 240

gataggccgt ttgtatgtgc accctcttca aaaactgggt ctgtgactac aacatattgc 300

tgcaatcagg accattgcaa taaaatagaa cttccaacta ctgtaaagtc atcacctggc 360

cttggtcctg tggaactggc agctgtcatt gctggaccag tgtgcttcgt ctgcatctca 420

ctcatgttga tggtctatat cagggccgca cggcacctgt gcccgccgct gcccacaccc 480

tgtgccagct ccgccattga gttccctgga gggaaggaga cttggcagtg gatcaaccca 540

gtggacttcc aggaagaggc atccctgcag gaggccctgg tggtagagat gtcctgggac 600

aaaggcgaga ggactgagcc tctcgagaag acagagctac ctgagggtgc ccctgagctg 660

gccctggata cagagttgtc cttggaggat ggagacaggt gcaaggccaa gatg 714

<210> 125

<211> 403

<212> PRT

<213> Artificial sequence

<220>

<223> TGFBR-IL12RB2 receptor

<400> 125

Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile Val Leu

1 5 10 15

Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val Gln Lys Ser Val

20 25 30

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

35 40 45

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

50 55 60

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

65 70 75 80

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

85 90 95

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

100 105 110

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

115 120 125

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

130 135 140

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu

145 150 155 160

Leu Leu Val Ile Phe Gln Val Thr Gly Ile Ser Leu Leu Pro Pro Leu

165 170 175

Gly Val Ala Ile Ser Val Ile Ile Ile Phe Tyr Gln Gln Lys Val Phe

180 185 190

Val Leu Leu Ala Ala Leu Arg Pro Gln Trp Cys Ser Arg Glu Ile Pro

195 200 205

Asp Pro Ala Asn Ser Thr Cys Ala Lys Lys Tyr Pro Ile Ala Glu Glu

210 215 220

Lys Thr Gln Leu Pro Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr Pro

225 230 235 240

Glu Asp Pro Glu Pro Leu Val Ile Ser Glu Val Leu His Gln Val Thr

245 250 255

Pro Val Phe Arg His Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu Lys

260 265 270

Gly Ile Gln Gly His Gln Ala Ser Glu Lys Asp Met Met His Ser Ala

275 280 285

Ser Ser Pro Pro Pro Pro Arg Ala Leu Gln Ala Glu Ser Arg Gln Leu

290 295 300

Val Asp Leu Tyr Lys Val Leu Glu Ser Arg Gly Ser Asp Pro Lys Pro

305 310 315 320

Glu Asn Pro Ala Cys Pro Trp Thr Val Leu Pro Ala Gly Asp Leu Pro

325 330 335

Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp Asp Leu Pro Ser His

340 345 350

Glu Ala Pro Leu Ala Asp Ser Leu Glu Glu Leu Glu Pro Gln His Ile

355 360 365

Ser Leu Ser Val Phe Pro Ser Ser Ser Leu His Pro Leu Thr Phe Ser

370 375 380

Cys Gly Asp Lys Leu Thr Leu Asp Gln Leu Lys Met Arg Cys Asp Ser

385 390 395 400

Leu Met Leu

<210> 126

<211> 1209

<212> DNA

<213> Artificial sequence

<220>

<223> TGFBR-IL12RB2 receptor

<400> 126

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ccagcaaaag gtgtttgttc tcctagcagc cctcagacct 600

cagtggtgta gcagagaaat tccagatcca gcaaatagca cttgcgctaa gaaatatccc 660

attgcagagg agaagacaca gctgcccttg gacaggctcc tgatagactg gcccacgcct 720

gaagatcctg aaccgctggt catcagtgaa gtccttcatc aagtgacccc agttttcaga 780

catcccccct gctccaactg gccacaaagg gaaaaaggaa tccaaggtca tcaggcctct 840

gagaaagaca tgatgcacag tgcctcaagc ccaccacctc caagagctct ccaagctgag 900

agcagacaac tggtggatct gtacaaggtg ctggagagca ggggctccga cccaaagcca 960

gaaaacccag cctgtccctg gacggtgctc ccagcaggtg accttcccac ccatgatggc 1020

tacttaccct ccaacataga tgacctcccc tcacatgagg cacctctcgc tgactctctg 1080

gaagaactgg agcctcagca catctccctt tctgttttcc cctcaagttc tcttcaccca 1140

ctcaccttct cctgtggtga taagctgact ctggatcagt taaagatgag gtgtgactcc 1200

ctcatgctc 1209

<210> 127

<211> 268

<212> PRT

<213> Artificial sequence

<220>

<223> TIM3-CD28 receptor

<400> 127

Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln

20 25 30

Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu

35 40 45

Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly

50 55 60

Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser

65 70 75 80

Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr

85 90 95

Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile

100 105 110

Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val

115 120 125

Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe

130 135 140

Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala

145 150 155 160

Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile

165 170 175

Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu

180 185 190

Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu Val Val Val Gly

195 200 205

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

210 215 220

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

225 230 235 240

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

245 250 255

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

260 265

<210> 128

<211> 804

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28 receptor

<400> 128

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctcc 804

<210> 129

<211> 503

<212> PRT

<213> Artificial sequence

<220>

<223> 13G4-1211 PD-L1/CD28 bispecific antibody

<400> 129

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala

20 25 30

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile

35 40 45

Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser

100 105 110

Tyr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Ser Gly

115 120 125

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val

130 135 140

Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr

145 150 155 160

Phe Asp Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly

165 170 175

Leu Glu Trp Val Ser Gly Ile Ser Trp Asn Arg Gly Arg Ile Glu Tyr

180 185 190

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

195 200 205

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

210 215 220

Leu Tyr Tyr Cys Ala Lys Gly Arg Phe Arg Tyr Phe Asp Trp Phe Leu

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

260 265 270

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

275 280 285

Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

290 295 300

Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu

305 310 315 320

Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr

325 330 335

Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr

340 345 350

Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp

355 360 365

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln

385 390 395 400

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

405 410 415

Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp

420 425 430

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala

435 440 445

Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

450 455 460

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

465 470 475 480

Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly

485 490 495

Gly Gly Thr Lys Val Glu Ile

500

<210> 130

<211> 1508

<212> DNA

<213> Artificial sequence

<220>

<223> 13G4-1211 PD-L1/CD28 bispecific antibody

<400> 130

atggggtggt cgtgtatcat cctgttcctg gtcgcgacag caaccggcgt gcattcggcc 60

atacagctga cccagagccc ctcctccctc tccgcttccg tgggggaccg cgtgacaatc 120

acgtgccgcg ccagccaggg aatctcctcg gccctcgcct ggtaccagca gaaacccggg 180

aaggctccca agctgctcat ctacgatgcc tcctcgcttg agtcgggcgt gccatccagg 240

ttctccggat ccgggtccgg aaccgacttt acactcacga tttcctctct gcagcccgag 300

gacttcgcca catactactg tcagcagttc aactcctacc cattcacctt cggcccgggc 360

accaaggtgg acatcaagtc tggcggggga ggctccgaag tccagctcgt ggaatccggg 420

ggcggtctcg tgcagccagg ccggagtctg cgcctgtctt gcgctgcctc ggggatcact 480

ttcgacgact acggcatgca ttgggttcgc caggccccag ggaaggggtt ggagtgggtc 540

agtggcattt catggaacag ggggcgcatc gaatacgccg actccgttaa gggcagattc 600

accatctcgc gcgataacgc caaaaacagt ctctacctcc agatgaactc gcttcgagca 660

gaggatactg ccctgtacta ttgcgcgaag ggacgcttcc gctactttga ctggtttctg 720

gactactggg gccaggggac actggtgacg gtgtcgtcgg ggggcggggg gagtcaggtg 780

cagctggtgc agtccggagc cgaggtaaag aagccaggcg cttccgtcaa ggtgtcatgc 840

aaggcctcag gctacacctt cacaagctat tacatccact gggtgcgcca agctcccggt 900

cagggcttgg agtggatcgg gtgcatttac ccagggaacg tcaacacaaa ctacaacgag 960

aagttcaagg atcgggcaac cctgaccgtg gacacatcca tctctaccgc ctacatggag 1020

ctgtcacgcc tgcgctctga tgacaccgca gtgtacttct gtaccaggag tcactacggc 1080

ctggactgga actttgatgt ctggggccag ggaaccaccg tgacggtgtc cagtgtggag 1140

ggcggtagtg gcggctctgg tgggtccgga ggctcaggcg gcgtgatgga tgacattcag 1200

atgacccaga gtccctcctc cctctccgct tccgtcggag accgcgtgac catcacttgt 1260

cacgcctcac agaatatcta cgtgtggctg aactggtacc aacagaagcc cggcaaggcc 1320

cccaagctgc ttatctataa agcgtccaac ctccacacgg gagtcccttc ccgcttctcc 1380

ggatccggca gtgggacgga cttcacactc acaatctcgt cgctgcagcc agaggacttt 1440

gcgacgtact actgccagca gggccagacc tacccatata ctttcggcgg cgggaccaag 1500

gtggagat 1508

<210> 131

<211> 500

<212> PRT

<213> Artificial sequence

<220>

<223> 10A5-1412 PD-L1/CD28 bispecific antibody

<400> 131

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala

20 25 30

Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile

35 40 45

Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys

50 55 60

Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser

100 105 110

Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Gly

115 120 125

Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys

130 135 140

Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr

145 150 155 160

Phe Thr Ser Tyr Asp Val His Trp Val Arg Gln Ala Pro Gly Gln Arg

165 170 175

Leu Glu Trp Met Gly Trp Leu His Ala Asp Thr Gly Ile Thr Lys Phe

180 185 190

Ser Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala

195 200 205

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala

210 215 220

Val Tyr Tyr Cys Ala Arg Glu Arg Ile Gln Leu Trp Phe Asp Tyr Trp

225 230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gln

245 250 255

Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser

260 265 270

Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr

275 280 285

Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly

290 295 300

Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe Lys

305 310 315 320

Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr Met

325 330 335

Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Thr

340 345 350

Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Gln Gly

355 360 365

Thr Thr Val Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln Met Thr Gln

385 390 395 400

Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

405 410 415

Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp Tyr Gln Gln

420 425 430

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Asn Leu

435 440 445

His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

450 455 460

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

465 470 475 480

Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly Gly Gly Thr

485 490 495

Lys Val Glu Ile

500

<210> 132

<211> 1499

<212> DNA

<213> Artificial sequence

<220>

<223> 10A5-1412 PD-L1/CD28 bispecific antibody

<400> 132

atgggctgga gttgcatcat tctcttcctc gtggcgaccg caacaggggt gcactccgac 60

atccagatga cccagtcccc gagttccctg tctgcttccg tgggagatcg cgtgactatc 120

acctgccggg cttcccaggg catctcttcc tggctggcgt ggtaccagca gaaaccagaa 180

aaggctccta agtccctgat ctacgcagct tcgtccctcc aatccggcgt cccctctcgc 240

ttctccggct ccggatccgg caccgacttc acgctgacaa tctcgagttt gcagcccgag 300

gacttcgcca cctactactg ccagcagtac aactcctacc cttacacctt cggccagggc 360

acaaagctcg aaatcaagtc gggggggggc gggtcgcagg tccagctggt gcagtccggc 420

gccgaagtca agaagcccgg agcaagtgtg aaagtgtcgt gcaaggcaag tgggtatacc 480

ttcacctcat acgacgtaca ctgggtgcgc caggcgcccg gtcagcgcct tgagtggatg 540

ggctggctcc acgccgacac cggcattacc aagttctctc agaagttcca gggaagagtg 600

accataacac gcgacaccag tgcttccaca gcttacatgg aactttcgag tctgagatcc 660

gaggacacag ccgtgtatta ctgtgcccgt gagcgcatcc agctgtggtt cgactactgg 720

gggcagggca ccctcgtgac ggtgtcgtcg gggggcgggg ggagtcaggt gcagctggtg 780

cagtccggag ccgaggtaaa gaagccaggc gcttccgtca aggtgtcatg caaggcctca 840

ggctacacct tcacaagcta ttacatccac tgggtgcgcc aagctcccgg tcagggcttg 900

gagtggatcg ggtgcattta cccagggaac gtcaacacaa actacaacga gaagttcaag 960

gatcgggcaa ccctgaccgt ggacacatcc atctctaccg cctacatgga gctgtcacgc 1020

ctgcgctctg atgacaccgc agtgtacttc tgtaccagga gtcactacgg cctggactgg 1080

aactttgatg tctggggcca gggaaccacc gtgacggtgt ccagtgtgga gggcggtagt 1140

ggcggctctg gtgggtccgg aggctcaggc ggcgtgatgg atgacattca gatgacccag 1200

agtccctcct ccctctccgc ttccgtcgga gaccgcgtga ccatcacttg tcacgcctca 1260

cagaatatct acgtgtggct gaactggtac caacagaagc ccggcaaggc ccccaagctg 1320

cttatctata aagcgtccaa cctccacacg ggagtccctt cccgcttctc cggatccggc 1380

agtgggacgg acttcacact cacaatctcg tcgctgcagc cagaggactt tgcgacgtac 1440

tactgccagc agggccagac ctacccatat actttcggcg gcgggaccaa ggtggagat 1499

<210> 133

<211> 507

<212> PRT

<213> Artificial sequence

<220>

<223> 1B12-1412 PD-L1/CD28 bispecific antibody

<400> 133

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn

100 105 110

Trp Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Gly Gly

115 120 125

Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

130 135 140

Pro Gly Ser Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe

145 150 155 160

Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

165 170 175

Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Arg Ala His Tyr Ala

180 185 190

Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser

195 200 205

Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val

210 215 220

Tyr Phe Cys Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly

225 230 235 240

Met Asp Val Trp Gly Gln Gly Thr Val Thr Val Ser Ser Gly Gly Ser

245 250 255

Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu

260 265 270

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

275 280 285

Tyr Thr Phe Thr Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly

290 295 300

Gln Gly Leu Glu Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr

305 310 315 320

Asn Tyr Asn Glu Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr

325 330 335

Ser Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp

340 345 350

Thr Ala Val Tyr Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn

355 360 365

Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu

370 375 380

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met

385 390 395 400

Asp Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

405 410 415

Gly Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val

420 425 430

Trp Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

435 440 445

Ile Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser

450 455 460

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

465 470 475 480

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro

485 490 495

Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

500 505

<210> 134

<211> 1520

<212> DNA

<213> Artificial sequence

<220>

<223> 1B12-1412 PD-L1/CD28 bispecific antibody

<400> 134

atgggctgga gttgcatcat cctctttcta gtcgccacgg ccaccggcgt acactcagag 60

atcgtgctga cacagtcgcc tgcgacgctg tcgctcagtc caggggagcg cgctactctc 120

tcctgccgcg cgtcgcagag cgtgtcgtcc tacttggcct ggtaccagca gaagcctggc 180

caggctccgc gcctgctgat atacgacgcc tcgaacagag ccacgggcat ccccgcccgt 240

tttagtggct ccgggtcggg gaccgacttc actctgacaa tctcatccct cgagcccgag 300

gatttcgccg tgtactactg tcagcagcgc tcgaattggc caaccttcgg gcaggggacg 360

aaagttgaga tcaaaagcgg cggcgggggc agccaggtcc agctcgtcca gtctggcgcc 420

gaggtcaaaa agccgggctc ttcggtcaag gtctcctgca agacttccgg cgacaccttc 480

tcctcctatg ctatctcctg ggtgcggcag gccccggggc agggcctgga gtggatggga 540

ggcatcatcc caatctttgg gagggcccac tacgcccaga agttccaggg acgcgtgaca 600

atcaccgcag acgagtccac atccactgcc tacatggagt tgtcctcgct ccggtcggag 660

gatactgccg tgtacttctg cgcccggaag ttccacttcg tgtcaggctc ccccttcggg 720

atggacgtgt ggggacaagg aaccgtgacg gtgtcgtcgg ggggctcgtc ggggggcggg 780

gggagtcagg tgcagctggt gcagtccgga gccgaggtaa agaagccagg cgcttccgtc 840

aaggtgtcat gcaaggcctc aggctacacc ttcacaagct attacatcca ctgggtgcgc 900

caagctcccg gtcagggctt ggagtggatc gggtgcattt acccagggaa cgtcaacaca 960

aactacaacg agaagttcaa ggatcgggca accctgaccg tggacacatc catctctacc 1020

gcctacatgg agctgtcacg cctgcgctct gatgacaccg cagtgtactt ctgtaccagg 1080

agtcactacg gcctggactg gaactttgat gtctggggcc agggaaccac cgtgacggtg 1140

tccagtgtgg agggcggtag tggcggctct ggtgggtccg gaggctcagg cggcgtgatg 1200

gatgacattc agatgaccca gagtccctcc tccctctccg cttccgtcgg agaccgcgtg 1260

accatcactt gtcacgcctc acagaatatc tacgtgtggc tgaactggta ccaacagaag 1320

cccggcaagg cccccaagct gcttatctat aaagcgtcca acctccacac gggagtccct 1380

tcccgcttct ccggatccgg cagtgggacg gacttcacac tcacaatctc gtcgctgcag 1440

ccagaggact ttgcgacgta ctactgccag cagggccaga cctacccata tactttcggc 1500

ggcgggacca aggtggagat 1520

<210> 135

<211> 504

<212> PRT

<213> Artificial sequence

<220>

<223> TGFBR-1-1412 TGFBRII/CD28 bispecific antibody

<400> 135

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Arg Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn

100 105 110

Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Gly

115 120 125

Gly Gly Gly Ser Gln Leu Gln Val Gln Glu Ser Gly Pro Gly Leu Val

130 135 140

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser

145 150 155 160

Ile Ser Asn Ser Tyr Phe Ser Trp Gly Trp Ile Arg Gln Pro Pro Gly

165 170 175

Lys Gly Leu Glu Trp Ile Gly Ser Phe Tyr Tyr Gly Glu Lys Thr Tyr

180 185 190

Tyr Asn Pro Ser Leu Lys Ser Arg Ala Thr Ile Ser Ile Asp Thr Ser

195 200 205

Lys Ser Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr

210 215 220

Ala Val Tyr Tyr Cys Pro Arg Gly Pro Thr Met Ile Arg Gly Val Ile

225 230 235 240

Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

260 265 270

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

275 280 285

Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

290 295 300

Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu

305 310 315 320

Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr

325 330 335

Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr

340 345 350

Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp

355 360 365

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln

385 390 395 400

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

405 410 415

Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp

420 425 430

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala

435 440 445

Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

450 455 460

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

465 470 475 480

Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly

485 490 495

Gly Gly Thr Lys Val Glu Ile Lys

500

<210> 136

<211> 1512

<212> DNA

<213> Artificial sequence

<220>

<223> TGFBR-1-1412 TGFBRII/CD28 bispecific antibody

<400> 136

atgggttggt cctgcatcat cctgtttctc gtggccaccg ccaccggcgt gcactccgaa 60

attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 120

tcctgcaggg ccagtcagag tgttcgcagc tacttagcct ggtaccaaca gaaacctggc 180

caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg 240

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 300

gattttgcag tttattactg tcagcagcgt agcaactggc ctccgacgtt cggccaaggg 360

accaaggtgg aaatcaaaag tggagggggc ggttcacagc tgcaggtgca ggagtcgggc 420

ccaggactgg tgaagccttc ggagaccctg tccctcacct gcactgtctc tggtggctcc 480

atcagcaaca gttatttctc ctggggctgg atccgccagc ccccagggaa gggactggag 540

tggattggga gtttctatta tggtgaaaaa acctactaca acccgtccct caagagccga 600

gccaccatat ccattgacac gtccaagagc cagttctccc tgaagctgag ctctgtgacc 660

gccgcagaca cggctgtgta ttactgtccg agagggccta ctatgattcg gggagttata 720

gactcctggg gccagggaac cctggtgacg gtgtcgtcgg ggggcggggg gagtcaggtg 780

cagctggtgc agtccggagc cgaggtaaag aagccaggcg cttccgtcaa ggtgtcatgc 840

aaggcctcag gctacacctt cacaagctat tacatccact gggtgcgcca agctcccggt 900

cagggcttgg agtggatcgg gtgcatttac ccagggaacg tcaacacaaa ctacaacgag 960

aagttcaagg atcgggcaac cctgaccgtg gacacatcca tctctaccgc ctacatggag 1020

ctgtcacgcc tgcgctctga tgacaccgca gtgtacttct gtaccaggag tcactacggc 1080

ctggactgga actttgatgt ctggggccag ggaaccaccg tgacggtgtc cagtgtggag 1140

ggcggtagtg gcggctctgg tgggtccgga ggctcaggcg gcgtgatgga tgacattcag 1200

atgacccaga gtccctcctc cctctccgct tccgtcggag accgcgtgac catcacttgt 1260

cacgcctcac agaatatcta cgtgtggctg aactggtacc aacagaagcc cggcaaggcc 1320

cccaagctgc ttatctataa agcgtccaac ctccacacgg gagtcccttc ccgcttctcc 1380

ggatccggca gtgggacgga cttcacactc acaatctcgt cgctgcagcc agaggacttt 1440

gcgacgtact actgccagca gggccagacc tacccatata ctttcggcgg cgggaccaag 1500

gtggagatta ag 1512

<210> 137

<211> 504

<212> PRT

<213> Artificial sequence

<220>

<223> TGFBR-3-1412 TGFBRII/CD28 bispecific antibody

<400> 137

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Arg Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn

100 105 110

Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Gly

115 120 125

Gly Gly Gly Ser Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

130 135 140

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser

145 150 155 160

Ile Ser Ser Ser Ser Tyr Ser Trp Gly Trp Ile Arg Gln Pro Pro Gly

165 170 175

Lys Gly Leu Glu Trp Ile Gly Ser Phe Tyr Tyr Ser Gly Ile Thr Tyr

180 185 190

Tyr Ser Pro Ser Leu Lys Ser Arg Ile Ile Ile Ser Glu Asp Thr Ser

195 200 205

Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr

210 215 220

Ala Val Tyr Tyr Cys Ala Ser Gly Phe Thr Met Ile Arg Gly Ala Leu

225 230 235 240

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

245 250 255

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

260 265 270

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

275 280 285

Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

290 295 300

Trp Ile Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu

305 310 315 320

Lys Phe Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Ile Ser Thr

325 330 335

Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr

340 345 350

Phe Cys Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp

355 360 365

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Val Glu Gly Gly Ser Gly

370 375 380

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Met Asp Asp Ile Gln

385 390 395 400

Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

405 410 415

Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp Leu Asn Trp

420 425 430

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala

435 440 445

Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

450 455 460

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

465 470 475 480

Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr Thr Phe Gly

485 490 495

Gly Gly Thr Lys Val Glu Ile Lys

500

<210> 138

<211> 1512

<212> DNA

<213> Artificial sequence

<220>

<223> TGFBR-3-1412 TGFBRII/CD28 bispecific antibody

<400> 138

atgggttggt cctgcatcat cctgtttctc gtggccaccg ccaccggcgt gcactccgaa 60

attgtgttga cacagtctcc agccaccctg tctttgtctc caggggaaag agccaccctc 120

tcctgcaggg ccagtcagag tgttagaagt ttcttagcct ggtaccaaca gaaacctggc 180

caggctccca ggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg 240

ttcagtggca gtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 300

gattttgcag tttattactg tcagcagcgt agcaactggc ctccgacgtt cggccaaggg 360

accaaggtgg aaatcaaaag tggagggggc ggttcacagc tacagctgca ggagtcgggc 420

ccaggactgg tgaagccttc ggagacccta tccctcacct gcactgtctc tggtggctcc 480

atcagcagta gtagttactc ctggggctgg atccgccagc ccccagggaa gggcctggag 540

tggattggga gtttctatta cagtgggatc acctactaca gcccgtccct caagagtcga 600

attatcatat ccgaagacac gtccaagaac cagttctccc tgaagctgag ttctgtgacc 660

gccgcagaca cggctgtgta ttactgtgcg agcgggttta ctatgattcg gggagccctt 720

gactactggg gccagggaac cctggtgacg gtgtcgtcgg ggggcggggg gagtcaggtg 780

cagctggtgc agtccggagc cgaggtaaag aagccaggcg cttccgtcaa ggtgtcatgc 840

aaggcctcag gctacacctt cacaagctat tacatccact gggtgcgcca agctcccggt 900

cagggcttgg agtggatcgg gtgcatttac ccagggaacg tcaacacaaa ctacaacgag 960

aagttcaagg atcgggcaac cctgaccgtg gacacatcca tctctaccgc ctacatggag 1020

ctgtcacgcc tgcgctctga tgacaccgca gtgtacttct gtaccaggag tcactacggc 1080

ctggactgga actttgatgt ctggggccag ggaaccaccg tgacggtgtc cagtgtggag 1140

ggcggtagtg gcggctctgg tgggtccgga ggctcaggcg gcgtgatgga tgacattcag 1200

atgacccaga gtccctcctc cctctccgct tccgtcggag accgcgtgac catcacttgt 1260

cacgcctcac agaatatcta cgtgtggctg aactggtacc aacagaagcc cggcaaggcc 1320

cccaagctgc ttatctataa agcgtccaac ctccacacgg gagtcccttc ccgcttctcc 1380

ggatccggca gtgggacgga cttcacactc acaatctcgt cgctgcagcc agaggacttt 1440

gcgacgtact actgccagca gggccagacc tacccatata ctttcggcgg cgggaccaag 1500

gtggagatta ag 1512

<210> 139

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 139

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 140

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> T2A

<400> 140

gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210> 141

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> spacer

<400> 141

Ser Gly Arg Ser Gly Gly Gly

1 5

<210> 142

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> spacer

<400> 142

tccggaagat ctggcggcgg a 21

<210> 143

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> F2A

<400> 143

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val

1 5 10 15

Glu Ser Asn Pro Gly Pro

20

<210> 144

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> F2A

<400> 144

gtgaaacaga ctttgaattt tgaccttctc aagttggcgg gagacgtgga gtccaaccca 60

gggccg 66

<210> 145

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> furin cleavage site

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<400> 145

Arg Xaa Lys Arg

1

<210> 146

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> furin cleavage site

<220>

<221> misc_feature

<222> (2)..(2)

<223> Xaa can be any naturally occurring amino acid

<400> 146

Arg Xaa Arg Arg

1

<210> 147

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> furin cleavage site

<220>

<221> misc_feature

<222> (2)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 147

Arg Xaa Xaa Arg

1

<210> 148

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> furin cleavage site

<400> 148

Arg Gln Lys Arg

1

<210> 149

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> furin cleavage site

<220>

<221> misc_feature

<222> (1)..(1)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (3)..(4)

<223> Xaa can be any naturally occurring amino acid

<400> 149

Xaa Arg Xaa Xaa Arg

1 5

<210> 150

<211> 96

<212> DNA

<213> Artificial sequence

<220>

<223> F-GS2-T2A linker

<400> 150

cgtgcgaaga ggggcggcgg gggctccggc gggggaggca gtgagggccg cggctccctg 60

ctgacctgcg gagatgtaga agagaaccca ggcccc 96

<210> 151

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> F-GS2-T2A linker

<400> 151

Arg Ala Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gly

1 5 10 15

Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro

20 25 30

<210> 152

<211> 2142

<212> DNA

<213> Artificial sequence

<220>

<223> TGFbRDN-PSMA-CAR

<400> 152

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac catggccctg cctgtgacag ccctgctgct gcctctggct 720

ctgctgctgc acgccgccag acctggatct gacattgtga tgacccagtc tcacaaattc 780

atgtccacat cagtaggaga cagggtcagc atcatctgta aggccagtca agatgtgggt 840

actgctgtag actggtatca acagaaacca ggacaatctc ctaaactact gatttattgg 900

gcatccactc ggcacactgg agtccctgat cgcttcacag gcagtggatc tgggacagac 960

ttcactctca ccattactaa cgttcagtct gaagacttgg cagattattt ctgtcagcaa 1020

tataacagct atcctctcac gttcggtgct gggaccatgc tggacctgaa aggaggcgga 1080

ggatctggcg gcggaggaag ttctggcgga ggcagcgagg tgcagctgca gcagagcgga 1140

cccgagctcg tgaagcctgg aacaagcgtg cggatcagct gcaagaccag cggctacacc 1200

ttcaccgagt acaccatcca ctgggtcaag cagtcccacg gcaagagcct ggagtggatc 1260

ggcaatatca accccaacaa cggcggcacc acctacaacc agaagttcga ggacaaggcc 1320

accctgaccg tggacaagag cagcagcacc gcctacatgg aactgcggag cctgaccagc 1380

gaggacagcg ccgtgtacta ttgtgccgcc ggttggaact tcgactactg gggccagggc 1440

acaaccctga cagtgtctag cgctagctcc ggaaccacga cgccagcgcc gcgaccacca 1500

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1560

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1620

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1680

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1740

actactcaag aggaagacgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1800

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 1860

cagctctata acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga 1920

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1980

tacaacgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 2040

gagcgccgga ggggcaaggg gcacgacggc ctttaccagg gtctcagtac agccaccaag 2100

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 2142

<210> 153

<211> 2151

<212> DNA

<213> Artificial sequence

<220>

<223> TGFbRDN-1C3PSMA-CAR

<400> 153

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc 720

ttgctgctcc acgccgccag gccgcaggtg caactggtgg agtctggggg aggcgtggtc 780

cagcctggga ggtccctgag actctcctgt gcagcctctg gattcacctt cagtagctat 840

gctatgcact gggtccgcca ggctccaggc aaggggctgg agtgggtggc agttatatca 900

tatgatggaa acaataaata ctacgcagac tccgtgaagg gccgattcac catctccaga 960

gacaattcca agaacacgct gtatctgcaa atgaacagcc tgagagctga ggacacggct 1020

gtgtattact gtgcgagagc cgtcccctgg ggatcgaggt actactacta cggtatggac 1080

gtctggggcc aagggaccac ggtcaccgtc tcctcaggtg gcggtggctc gggcggtggt 1140

gggtcgggtg gcggcggatc tgccatccag ttgacccagt ctccatcctc cctgtctgca 1200

tctgtaggag acagagtcac catcacttgc cgggcaagtc agggcattag cagtgcttta 1260

gcctggtatc agcagaaatc agggaaagct cctaagctcc tgatctttga tgcctccagt 1320

ttggaaagtg gggtcccatc aaggttcagc ggcagtggat ctgggacaga tttcactctc 1380

accatcagca gcctgcagcc tgaagatttt gcaacttatt actgtcaaca gtttaacagt 1440

tatcctctca ctttcggcgg agggaccaag gtggagatca aaaccacgac gccagcgccg 1500

cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1560

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 1620

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1680

ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1740

ccagtacaaa ctactcaaga ggaagacggc tgtagctgcc gatttccaga agaagaagaa 1800

ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 1860

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgacgttttg 1920

gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1980

gaaggcctgt acaacgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 2040

atgaaaggcg agcgccggag gggcaagggg cacgacggcc tttaccaggg tctcagtaca 2100

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg c 2151

<210> 154

<211> 2136

<212> DNA

<213> Artificial sequence

<220>

<223> TGFbRDN-2A10PSMA-CAR

<400> 154

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc 720

ttgctgctcc acgccgccag gccggaggtg cagctggtgc agtctggagc agaggtgaaa 780

aagcccgggg agtctctgaa gatctcctgt aagggttctg gatacagctt taccagtaac 840

tggatcggct gggtgcgcca gatgcccggg aaaggcctgg agtggatggg gatcatctat 900

cctggtgact ctgataccag atacagcccg tccttccaag gccaggtcac catctcagcc 960

gacaagtcca tcagcaccgc ctacctgcag tggagcagcc tgaaggcctc ggacaccgcc 1020

atgtattact gtgcgaggca aactggtttc ctctggtcct ccgatctctg gggccgtggc 1080

accctggtca ctgtctcctc aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 1140

ggatctgcca tccagttgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 1200

gtcaccatca cttgccgggc aagtcaggac attagcagtg ctttagcctg gtatcaacag 1260

aaaccaggga aagctcctaa gctcctgatc tatgatgcct ccagtttgga aagtggggtc 1320

ccatcaaggt tcagcggcta tggatctggg acagatttca ctctcaccat caacagcctg 1380

cagcctgaag attttgcaac ttattactgt caacagttta atagttaccc gctcactttc 1440

ggcggaggga ccaaggtgga gatcaaaacc acgacgccag cgccgcgacc accaacaccg 1500

gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 1560

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 1620

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1680

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1740

caagaggaag acggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1800

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1860

tataacgagc tcaatctagg acgaagagag gagtacgacg ttttggacaa gagacgtggc 1920

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaac 1980

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 2040

cggaggggca aggggcacga cggcctttac cagggtctca gtacagccac caaggacacc 2100

tacgacgccc ttcacatgca ggccctgccc cctcgc 2136

<210> 155

<211> 2142

<212> DNA

<213> Artificial sequence

<220>

<223> TGFbRDN-2F5PSMA-CAR

<400> 155

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc 720

ttgctgctcc acgccgccag gccggaggtg cagctggtgc agtctggagc agaggtgaaa 780

aagcccgggg agtctctgaa gatctcctgt aagggttctg gatacagttt taccagcaac 840

tggatcggct gggtgcgcca gatgcccggg aaaggcctgg agtggatggg gatcatctat 900

cctggtgact ctgataccag atacagcccg tccttccaag gccaggtcac catctcagcc 960

gacaagtcca tcagcaccgc ctacctgcag tggaacagcc tgaaggcctc ggacaccgcc 1020

atgtattact gtgcgagaca aactggtttc ctctggtcct tcgatctctg gggccgtggc 1080

accctggtca ctgtctcctc aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 1140

ggatctgcca tccagttgac ccagtctcca tcctccctgt ctgcatctgt aggagacaga 1200

gtcaccatca cttgccgggc aagtcaggac attagcagtg ctttagcctg gtatcagcag 1260

aaaccgggga aagctcctaa gctcctgatc tatgatgcct ccagtttgga aagtggggtc 1320

ccatcaaggt tcagcggcag tggatctggg acagatttca ctctcaccat cagcagcctg 1380

cagcctgaag attttgcaac ttattactgt caacagttta atagttaccc gctcactttc 1440

ggcggaggga ccaaggtgga gatcaaaatc aaaaccacga cgccagcgcc gcgaccacca 1500

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1560

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1620

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1680

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1740

actactcaag aggaagacgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1800

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 1860

cagctctata acgagctcaa tctaggacga agagaggagt acgacgtttt ggacaagaga 1920

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1980

tacaacgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 2040

gagcgccgga ggggcaaggg gcacgacggc ctttaccagg gtctcagtac agccaccaag 2100

gacacctacg acgcccttca catgcaggcc ctgccccctc gc 2142

<210> 156

<211> 2145

<212> DNA

<213> Artificial sequence

<220>

<223> TGFbRDN-2C6PSMA-CAR

<400> 156

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac catggcctta ccagtgaccg ccttgctcct gccgctggcc 720

ttgctgctcc acgccgccag gccggaggtg cagctggtgc agtctggatc agaggtgaaa 780

aagcccgggg agtctctgaa gatctcctgt aagggttctg gatacagctt taccaactac 840

tggatcggct gggtgcgcca gatgcccggg aaaggcctgg agtggatggg gatcatctat 900

cctggtgact ctgataccag atacagcccg tccttccaag gccaggtcac catctcagcc 960

gacaagtcca tcagcaccgc ctatctgcag tggagcagcc tgaaggcctc ggacaccgcc 1020

atgtattact gtgcgagtcc cgggtatacc agcagttgga cttcttttga ctactggggc 1080

cagggaaccc tggtcaccgt ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt 1140

ggcggcggat ctgaaattgt gttgacacag tctccagcca ccctgtcttt gtctccaggg 1200

gaaagagcca ccctctcctg cagggccagt cagagtgtta gcagctactt agcctggtac 1260

caacagaaac ctggccaggc tcccaggctc ctcatctatg atgcatccaa cagggccact 1320

ggcatcccag ccaggttcag tggcagtggg tctgggacag acttcactct caccatcagc 1380

agcctagagc ctgaagattt tgcagtttat tactgtcagc agcgtagcaa ctggccccta 1440

ttcactttcg gccctgggac caaagtggat atcaaaacca cgacgccagc gccgcgacca 1500

ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 1560

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 1620

tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1680

tgcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1740

caaactactc aagaggaaga cggctgtagc tgccgatttc cagaagaaga agaaggagga 1800

tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 1860

aaccagctct ataacgagct caatctagga cgaagagagg agtacgacgt tttggacaag 1920

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1980

ctgtacaacg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 2040

ggcgagcgcc ggaggggcaa ggggcacgac ggcctttacc agggtctcag tacagccacc 2100

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 2145

<210> 157

<211> 2250

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28-1C3PSMA-CAR

<400> 157

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 900

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 960

gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa caataaatac 1020

tacgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 1080

tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc 1140

gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg 1200

gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 1260

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 1320

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaatca 1380

gggaaagctc ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca 1440

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 1500

gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga 1560

gggaccaagg tggagatcaa aaccacgacg ccagcgccgc gaccaccaac accggcgccc 1620

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 1680

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 1740

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1800

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1860

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1920

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1980

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 2040

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 2100

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 2160

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 2220

gcccttcaca tgcaggccct gccccctcgc 2250

<210> 158

<211> 2235

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28-2A10PSMA-CAR

<400> 158

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900

atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag 960

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 1020

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 1080

tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 1140

actggtttcc tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 1260

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320

agtcaggaca ttagcagtgc tttagcctgg tatcaacaga aaccagggaa agctcctaag 1380

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggctat 1440

ggatctggga cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact 1500

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 1560

atcaaaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 1620

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1680

gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1740

cttctcctgt cactggttat caccctttac tgcaaacggg gcagaaagaa actcctgtat 1800

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1860

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1920

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1980

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 2040

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 2100

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 2160

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 2220

gccctgcccc ctcgc 2235

<210> 159

<211> 2241

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28-2F5PSMA-CAR

<400> 159

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 960

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 1020

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 1080

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 1140

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 1260

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 1380

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 1440

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 1500

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 1560

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1680

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1740

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1800

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1860

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1920

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1980

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 2040

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 2100

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 2160

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 2220

atgcaggccc tgccccctcg c 2241

<210> 160

<211> 2244

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CTM-CD28-2C6PSMA-CAR

<400> 160

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccggaggtgc agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag 900

atctcctgta agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag 960

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 1020

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 1080

tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 1140

gggtatacca gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc 1200

tcctcaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaaattgtg 1260

ttgacacagt ctccagccac cctgtctttg tctccagggg aaagagccac cctctcctgc 1320

agggccagtc agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct 1380

cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc caggttcagt 1440

ggcagtgggt ctgggacaga cttcactctc accatcagca gcctagagcc tgaagatttt 1500

gcagtttatt actgtcagca gcgtagcaac tggcccctat tcactttcgg ccctgggacc 1560

aaagtggata tcaaaaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 1620

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 1680

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 1740

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1800

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1860

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1920

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1980

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 2040

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 2100

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 2160

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 2220

cacatgcagg ccctgccccc tcgc 2244

<210> 161

<211> 2232

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28-1C3PSMA-CAR

<400> 161

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccgcaggt gcaactggtg 840

gagtctgggg gaggcgtggt ccagcctggg aggtccctga gactctcctg tgcagcctct 900

ggattcacct tcagtagcta tgctatgcac tgggtccgcc aggctccagg caaggggctg 960

gagtgggtgg cagttatatc atatgatgga aacaataaat actacgcaga ctccgtgaag 1020

ggccgattca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 1080

ctgagagctg aggacacggc tgtgtattac tgtgcgagag ccgtcccctg gggatcgagg 1140

tactactact acggtatgga cgtctggggc caagggacca cggtcaccgt ctcctcaggt 1200

ggcggtggct cgggcggtgg tgggtcgggt ggcggcggat ctgccatcca gttgacccag 1260

tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 1320

cagggcatta gcagtgcttt agcctggtat cagcagaaat cagggaaagc tcctaagctc 1380

ctgatctttg atgcctccag tttggaaagt ggggtcccat caaggttcag cggcagtgga 1440

tctgggacag atttcactct caccatcagc agcctgcagc ctgaagattt tgcaacttat 1500

tactgtcaac agtttaacag ttatcctctc actttcggcg gagggaccaa ggtggagatc 1560

aaaaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 1620

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 1680

ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 1740

ctcctgtcac tggttatcac cctttactgc aaacggggca gaaagaaact cctgtatata 1800

ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1860

cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1920

gacgcccccg cgtacaagca gggccagaac cagctctata acgagctcaa tctaggacga 1980

agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 2040

ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 2100

gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 2160

ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 2220

ctgccccctc gc 2232

<210> 162

<211> 2217

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28-2A10PSMA-CAR

<400> 162

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt gcagctggtg 840

cagtctggag cagaggtgaa aaagcccggg gagtctctga agatctcctg taagggttct 900

ggatacagct ttaccagtaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg 960

gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc gtccttccaa 1020

ggccaggtca ccatctcagc cgacaagtcc atcagcaccg cctacctgca gtggagcagc 1080

ctgaaggcct cggacaccgc catgtattac tgtgcgaggc aaactggttt cctctggtcc 1140

tccgatctct ggggccgtgg caccctggtc actgtctcct caggtggcgg tggctcgggc 1200

ggtggtgggt cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg 1260

tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga cattagcagt 1320

gctttagcct ggtatcaaca gaaaccaggg aaagctccta agctcctgat ctatgatgcc 1380

tccagtttgg aaagtggggt cccatcaagg ttcagcggct atggatctgg gacagatttc 1440

actctcacca tcaacagcct gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500

aatagttacc cgctcacttt cggcggaggg accaaggtgg agatcaaaac cacgacgcca 1560

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 1620

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 1680

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1740

atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 1800

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1860

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1920

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1980

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 2040

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 2100

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 2160

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 2217

<210> 163

<211> 2223

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28-25FPSMA-CAR

<400> 163

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt gcagctggtg 840

cagtctggag cagaggtgaa aaagcccggg gagtctctga agatctcctg taagggttct 900

ggatacagtt ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg 960

gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc gtccttccaa 1020

ggccaggtca ccatctcagc cgacaagtcc atcagcaccg cctacctgca gtggaacagc 1080

ctgaaggcct cggacaccgc catgtattac tgtgcgagac aaactggttt cctctggtcc 1140

ttcgatctct ggggccgtgg caccctggtc actgtctcct caggtggcgg tggctcgggc 1200

ggtggtgggt cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg 1260

tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga cattagcagt 1320

gctttagcct ggtatcagca gaaaccgggg aaagctccta agctcctgat ctatgatgcc 1380

tccagtttgg aaagtggggt cccatcaagg ttcagcggca gtggatctgg gacagatttc 1440

actctcacca tcagcagcct gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500

aatagttacc cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg 1560

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 1620

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 1680

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 1740

ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1800

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1860

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1920

gcgtacaagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1980

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 2040

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2100

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 2160

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 2220

cgc 2223

<210> 164

<211> 2226

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM-CD28-2C6PSMA-CAR

<400> 164

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt gcagctggtg 840

cagtctggat cagaggtgaa aaagcccggg gagtctctga agatctcctg taagggttct 900

ggatacagct ttaccaacta ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg 960

gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc gtccttccaa 1020

ggccaggtca ccatctcagc cgacaagtcc atcagcaccg cctatctgca gtggagcagc 1080

ctgaaggcct cggacaccgc catgtattac tgtgcgagtc ccgggtatac cagcagttgg 1140

acttcttttg actactgggg ccagggaacc ctggtcaccg tctcctcagg tggcggtggc 1200

tcgggcggtg gtgggtcggg tggcggcgga tctgaaattg tgttgacaca gtctccagcc 1260

accctgtctt tgtctccagg ggaaagagcc accctctcct gcagggccag tcagagtgtt 1320

agcagctact tagcctggta ccaacagaaa cctggccagg ctcccaggct cctcatctat 1380

gatgcatcca acagggccac tggcatccca gccaggttca gtggcagtgg gtctgggaca 1440

gacttcactc tcaccatcag cagcctagag cctgaagatt ttgcagttta ttactgtcag 1500

cagcgtagca actggcccct attcactttc ggccctggga ccaaagtgga tatcaaaacc 1560

acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 1620

ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 1680

ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 1740

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 1800

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1860

ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 1920

cccgcgtaca agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1980

gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 2040

aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 2100

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 2160

cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 2220

cctcgc 2226

<210> 165

<211> 2340

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-1C3PSMA-CAR

<400> 165

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccgcaggtgc aactggtgga gtctggggga 960

ggcgtggtcc agcctgggag gtccctgaga ctctcctgtg cagcctctgg attcaccttc 1020

agtagctatg ctatgcactg ggtccgccag gctccaggca aggggctgga gtgggtggca 1080

gttatatcat atgatggaaa caataaatac tacgcagact ccgtgaaggg ccgattcacc 1140

atctccagag acaattccaa gaacacgctg tatctgcaaa tgaacagcct gagagctgag 1200

gacacggctg tgtattactg tgcgagagcc gtcccctggg gatcgaggta ctactactac 1260

ggtatggacg tctggggcca agggaccacg gtcaccgtct cctcaggtgg cggtggctcg 1320

ggcggtggtg ggtcgggtgg cggcggatct gccatccagt tgacccagtc tccatcctcc 1380

ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcaagtca gggcattagc 1440

agtgctttag cctggtatca gcagaaatca gggaaagctc ctaagctcct gatctttgat 1500

gcctccagtt tggaaagtgg ggtcccatca aggttcagcg gcagtggatc tgggacagat 1560

ttcactctca ccatcagcag cctgcagcct gaagattttg caacttatta ctgtcaacag 1620

tttaacagtt atcctctcac tttcggcgga gggaccaagg tggagatcaa aaccacgacg 1680

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 1740

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 1800

tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1860

gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1920

tttatgagac cagtacaaac tactcaagag gaagacggct gtagctgccg atttccagaa 1980

gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 2040

tacaagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 2100

gacgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 2160

aaccctcagg aaggcctgta caacgaactg cagaaagata agatggcgga ggcctacagt 2220

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgacggcct ttaccagggt 2280

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 2340

<210> 166

<211> 2325

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-2A10PSMA-CAR

<400> 166

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca 960

gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg atacagcttt 1020

accagtaact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 1140

atctcagccg acaagtccat cagcaccgcc tacctgcagt ggagcagcct gaaggcctcg 1200

gacaccgcca tgtattactg tgcgaggcaa actggtttcc tctggtcctc cgatctctgg 1260

ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg tggtgggtcg 1320

ggtggcggcg gatctgccat ccagttgacc cagtctccat cctccctgtc tgcatctgta 1380

ggagacagag tcaccatcac ttgccgggca agtcaggaca ttagcagtgc tttagcctgg 1440

tatcaacaga aaccagggaa agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500

agtggggtcc catcaaggtt cagcggctat ggatctggga cagatttcac tctcaccatc 1560

aacagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa tagttacccg 1620

ctcactttcg gcggagggac caaggtggag atcaaaacca cgacgccagc gccgcgacca 1680

ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga ggcgtgccgg 1740

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 1800

tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1860

tgcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1920

caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1980

tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 2040

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 2100

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 2160

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 2220

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 2280

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 2325

<210> 167

<211> 2331

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-25FPSMA-CAR

<400> 167

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca 960

gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg atacagtttt 1020

accagcaact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 1140

atctcagccg acaagtccat cagcaccgcc tacctgcagt ggaacagcct gaaggcctcg 1200

gacaccgcca tgtattactg tgcgagacaa actggtttcc tctggtcctt cgatctctgg 1260

ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg tggtgggtcg 1320

ggtggcggcg gatctgccat ccagttgacc cagtctccat cctccctgtc tgcatctgta 1380

ggagacagag tcaccatcac ttgccgggca agtcaggaca ttagcagtgc tttagcctgg 1440

tatcagcaga aaccggggaa agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500

agtggggtcc catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc 1560

agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa tagttacccg 1620

ctcactttcg gcggagggac caaggtggag atcaaaatca aaaccacgac gccagcgccg 1680

cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1740

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 1800

tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 1860

ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1920

ccagtacaaa ctactcaaga ggaagacggc tgtagctgcc gatttccaga agaagaagaa 1980

ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 2040

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgacgttttg 2100

gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 2160

gaaggcctgt acaacgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 2220

atgaaaggcg agcgccggag gggcaagggg cacgacggcc tttaccaggg tctcagtaca 2280

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg c 2331

<210> 168

<211> 2334

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-2C6PSMA-CAR

<400> 168

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggatca 960

gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg atacagcttt 1020

accaactact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 1140

atctcagccg acaagtccat cagcaccgcc tatctgcagt ggagcagcct gaaggcctcg 1200

gacaccgcca tgtattactg tgcgagtccc gggtatacca gcagttggac ttcttttgac 1260

tactggggcc agggaaccct ggtcaccgtc tcctcaggtg gcggtggctc gggcggtggt 1320

gggtcgggtg gcggcggatc tgaaattgtg ttgacacagt ctccagccac cctgtctttg 1380

tctccagggg aaagagccac cctctcctgc agggccagtc agagtgttag cagctactta 1440

gcctggtacc aacagaaacc tggccaggct cccaggctcc tcatctatga tgcatccaac 1500

agggccactg gcatcccagc caggttcagt ggcagtgggt ctgggacaga cttcactctc 1560

accatcagca gcctagagcc tgaagatttt gcagtttatt actgtcagca gcgtagcaac 1620

tggcccctat tcactttcgg ccctgggacc aaagtggata tcaaaaccac gacgccagcg 1680

ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 1740

gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1800

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1860

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1920

agaccagtac aaactactca agaggaagac ggctgtagct gccgatttcc agaagaagaa 1980

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 2040

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgacgtt 2100

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 2160

caggaaggcc tgtacaacga actgcagaaa gataagatgg cggaggccta cagtgagatt 2220

gggatgaaag gcgagcgccg gaggggcaag gggcacgacg gcctttacca gggtctcagt 2280

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 2334

<210> 169

<211> 821

<212> DNA

<213> Artificial sequence

<220>

<223> 1C3

<400> 169

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc aactggtgga gtctggggga ggcgtggtcc agcctgggag gtccctgaga 120

ctctcctgtg cagcctctgg attcaccttc agtagctatg ctatgcactg ggtccgccag 180

gctccaggca aggggctgga gtgggtggca gttatatcat atgatggaaa caataaatac 240

tacgcagact ccgtgaaggg ccgattcacc atctccagag acaattccaa gaacacgctg 300

tatctgcaaa tgaacagcct gagagctgag gacacggctg tgtattactg tgcgagagcc 360

gtcccctggg gatcgaggta ctactactac ggtatggacg tctggggcca agggaccacg 420

gtcaccgtct cctcaggtgg cggtggctcg ggcggtggtg ggtcgggtgg cggcggatct 480

gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 540

atcacttgcc gggcaagtca gggcattagc agtgctttag cctggtatca gcagaaatca 600

gggaaagctc ctaagctcct gatctttgat gcctccagtt tggaaagtgg ggtcccatca 660

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720

gaagattttg caacttatta ctgtcaacag tttaacagtt atcctctcac tttcggcgga 780

gggaccaagg tggagatcaa aaccacgacg ccagcgccgc g 821

<210> 170

<211> 806

<212> DNA

<213> Artificial sequence

<220>

<223> 2A10

<400> 170

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accagtaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgaggcaa 360

actggtttcc tctggtcctc cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcaacaga aaccagggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggctat 660

ggatctggga cagatttcac tctcaccatc aacagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaacca cgacgccagc gccgcg 806

<210> 171

<211> 812

<212> DNA

<213> Artificial sequence

<220>

<223> 2F5

<400> 171

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaatca aaaccacgac gccagcgccg cg 812

<210> 172

<211> 815

<212> DNA

<213> Artificial sequence

<220>

<223> 2C6

<400> 172

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggatca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagcttt accaactact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tatctgcagt ggagcagcct gaaggcctcg gacaccgcca tgtattactg tgcgagtccc 360

gggtatacca gcagttggac ttcttttgac tactggggcc agggaaccct ggtcaccgtc 420

tcctcaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaaattgtg 480

ttgacacagt ctccagccac cctgtctttg tctccagggg aaagagccac cctctcctgc 540

agggccagtc agagtgttag cagctactta gcctggtacc aacagaaacc tggccaggct 600

cccaggctcc tcatctatga tgcatccaac agggccactg gcatcccagc caggttcagt 660

ggcagtgggt ctgggacaga cttcactctc accatcagca gcctagagcc tgaagatttt 720

gcagtttatt actgtcagca gcgtagcaac tggcccctat tcactttcgg ccctgggacc 780

aaagtggata tcaaaaccac gacgccagcg ccgcg 815

<210> 173

<211> 780

<212> DNA

<213> Artificial sequence

<220>

<223> PD1.CD28-F2A

<400> 173

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

<210> 174

<211> 762

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-PTM.CD28-F2A

<400> 174

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cg 762

<210> 175

<211> 681

<212> DNA

<213> Artificial sequence

<220>

<223> dnTGFRBII-T2A

<400> 175

atgggtcggg ggctgctcag gggcctgtgg ccgctgcaca tcgtcctgtg gacgcgtatc 60

gccagcacga tcccaccgca cgttcagaag tcggttaata acgacatgat agtcactgac 120

aacaacggtg cagtcaagtt tccacaactg tgtaaatttt gtgatgtgag attttccacc 180

tgtgacaacc agaaatcctg catgagcaac tgcagcatca cctccatctg tgagaagcca 240

caggaagtct gtgtggctgt atggagaaag aatgacgaga acataacact agagacagtt 300

tgccatgacc ccaagctccc ctaccatgac tttattctgg aagatgctgc ttctccaaag 360

tgcattatga aggaaaaaaa aaagcctggt gagactttct tcatgtgttc ctgtagctct 420

gatgagtgca atgacaacat catcttctca gaagaatata acaccagcaa tcctgacttg 480

ttgctagtca tatttcaagt gacaggcatc agcctcctgc caccactggg agttgccata 540

tctgtcatca tcatcttcta ctgctaccgc gttaaccggc agcagaagct gagttcatcc 600

ggaagatctg gcggcggaga gggcagagga agtcttctaa catgcggtga cgtggaggag 660

aatcccggcc ctagagccac c 681

<210> 176

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 176

aggaagtctc aaagtgccct 20

<210> 177

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 177

gaacaacagc tgctccactc 20

<210> 178

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 178

gctacactga gcaccaggtg gtctc 25

<210> 179

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 179

cccagcagtg agggtctctc tcttc 25

<210> 180

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> J591 PSMA scFv

<400> 180

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcatctgta aggccagtca agatgtgggt actgctgtag actggtatca acagaaacca 120

ggacaatctc ctaaactact gatttattgg gcatccactc ggcacactgg agtccctgat 180

cgcttcacag gcagtggatc tgggacagac ttcactctca ccattactaa cgttcagtct 240

gaagacttgg cagattattt ctgtcagcaa tataacagct atcctctcac gttcggtgct 300

gggaccatgc tggacctgaa aggaggcgga ggatctggcg gcggaggaag ttctggcgga 360

ggcagcgagg tgcagctgca gcagagcgga cccgagctcg tgaagcctgg aacaagcgtg 420

cggatcagct gcaagaccag cggctacacc ttcaccgagt acaccatcca ctgggtcaag 480

cagtcccacg gcaagagcct ggagtggatc ggcaatatca accccaacaa cggcggcacc 540

acctacaacc agaagttcga ggacaaggcc accctgaccg tggacaagag cagcagcacc 600

gcctacatgg aactgcggag cctgaccagc gaggacagcg ccgtgtacta ttgtgccgcc 660

ggttggaact tcgactactg gggccagggc acaaccctga cagtgtctag c 711

<210> 181

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> J591 VL

<400> 181

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Ile Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ala Ile Thr Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 182

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> J591 VH

<400> 182

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

100 105 110

Val Ser Ser

115

<210> 183

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH consensus

<220>

<221> misc_feature

<222> (9)..(9)

<223> X is A or P

<220>

<221> misc_feature

<222> (11)..(11)

<223> X is V or L

<220>

<221> misc_feature

<222> (24)..(24)

<223> X is A or T

<220>

<221> misc_feature

<222> (38)..(38)

<223> X is R or K

<220>

<221> misc_feature

<222> (41)..(41)

<223> X is P or H

<220>

<221> misc_feature

<222> (55)..(55)

<223> X is N or Q

<220>

<221> misc_feature

<222> (68)..(68)

<223> X is V or A

<220>

<221> misc_feature

<222> (70)..(70)

<223> X is I or L

<400> 183

Glu Val Gln Leu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Xaa Gln Ala Xaa Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Xaa Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Xaa Thr Xaa Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 184

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL consensus

<220>

<221> misc_feature

<222> (3)..(3)

<223> X is Q or V

<220>

<221> misc_feature

<222> (10)..(10)

<223> X is T or F

<220>

<221> misc_feature

<222> (63)..(63)

<223> X is S or T

<220>

<221> misc_feature

<222> (80)..(80)

<223> X is P or S

<220>

<221> misc_feature

<222> (85)..(85)

<223> X is V or D

<220>

<221> misc_feature

<222> (87)..(87)

<223> X is Y or F

<220>

<221> misc_feature

<222> (103)..(103)

<223> X is K or M

<400> 184

Asp Ile Xaa Met Thr Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Xaa Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Xaa

65 70 75 80

Glu Asp Phe Ala Xaa Tyr Xaa Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Xaa Val Asp Ile Lys

100 105

<210> 185

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 185

Glu Val Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ala His Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 186

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 186

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Met Val Asp Ile Lys

100 105

<210> 187

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 187

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 188

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 188

Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 189

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 189

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Ala Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 190

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 190

Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 191

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 191

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 192

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 192

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 193

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 193

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Gln Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 194

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH consensus

<220>

<221> misc_feature

<222> (9)..(9)

<223> X is A or P

<220>

<221> misc_feature

<222> (11)..(11)

<223> X is V or L

<220>

<221> misc_feature

<222> (24)..(24)

<223> X is A or T

<220>

<221> misc_feature

<222> (38)..(38)

<223> X is R or K

<220>

<221> misc_feature

<222> (41)..(41)

<223> X is P or H

<220>

<221> misc_feature

<222> (55)..(55)

<223> X is N or Q

<220>

<221> misc_feature

<222> (68)..(68)

<223> X is V or A

<220>

<221> misc_feature

<222> (70)..(70)

<223> X is I or L

<220>

<221> misc_feature

<222> (86)..(86)

<223> X is L or P

<220>

<221> misc_feature

<222> (98)..(102)

<223> is AYWLF, GGWTF, or GAWTM

<400> 194

Glu Val Gln Leu Val Gln Ser Gly Xaa Glu Xaa Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Xaa Gln Ala Xaa Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Xaa Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Xaa Thr Xaa Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Xaa Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Xaa Xaa Xaa Xaa Xaa Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 195

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL consensus

<220>

<221> misc_feature

<222> (3)..(3)

<223> X is Q or V

<220>

<221> misc_feature

<222> (10)..(10)

<223> X is T or F

<220>

<221> misc_feature

<222> (63)..(63)

<223> X is S or T

<220>

<221> misc_feature

<222> (80)..(80)

<223> X is P or S

<220>

<221> misc_feature

<222> (85)..(85)

<223> X is V or D

<220>

<221> misc_feature

<222> (87)..(87)

<223> X is Y or F

<220>

<221> misc_feature

<222> (91)..(95)

<223> is FTRYP or YNAYS

<220>

<221> misc_feature

<222> (103)..(103)

<223> X is K or M

<400> 195

Asp Ile Xaa Met Thr Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Xaa Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Xaa

65 70 75 80

Glu Asp Phe Ala Xaa Tyr Xaa Cys Gln Gln Xaa Xaa Xaa Xaa Xaa Leu

85 90 95

Thr Phe Gly Gln Gly Thr Val Asp Ile Lys

100 105

<210> 196

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 196

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Tyr Trp Leu Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 197

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 197

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 198

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 198

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Gly Trp Thr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 199

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 199

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Thr Arg Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 200

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 200

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Ala Trp Thr Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 201

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VL

<400> 201

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ala Tyr Ser Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys

100 105

<210> 202

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> humanized PSMA VH

<400> 202

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Thr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Glu Asp Arg Val Thr Ile Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Pro Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

100 105 110

Val Ser Ser

115

<210> 203

<211> 35

<212> PRT

<213> Artificial sequence

<220>

<223> ICOS ICD

<400> 203

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 204

<211> 105

<212> DNA

<213> Artificial sequence

<220>

<223> ICOS ICD

<400> 204

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105

<210> 205

<211> 148

<212> PRT

<213> Artificial sequence

<220>

<223> ICOS CD3ζ ICD

<400> 205

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

35 40 45

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

50 55 60

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

65 70 75 80

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

85 90 95

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

100 105 110

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

115 120 125

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

130 135 140

Leu Pro Pro Arg

145

<210> 206

<211> 444

<212> DNA

<213> Artificial sequence

<220>

<223> ICOS CD3ζ ICD

<400> 206

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga ccctaagagt gaagttcagc 120

aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 180

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 240

gggggaaagc cgcagagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 300

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 360

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 420

cacatgcagg ccctgccccc tcgc 444

<210> 207

<211> 148

<212> PRT

<213> Artificial sequence

<220>

<223> variant ICOS CD3 ζ ICD

<400> 207

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

35 40 45

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

50 55 60

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

65 70 75 80

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

85 90 95

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

100 105 110

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

115 120 125

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

130 135 140

Leu Pro Pro Arg

145

<210> 208

<211> 444

<212> DNA

<213> Artificial sequence

<220>

<223> variant ICOS CD3 ζ ICD

<400> 208

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gaacatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga ccctaagagt gaagttcagc 120

aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 180

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 240

gggggaaagc cgcagagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 300

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 360

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 420

cacatgcagg ccctgccccc tcgc 444

<210> 209

<211> 481

<212> PRT

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR ICOS CD3z

<400> 209

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val

305 310 315 320

Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys

325 330 335

Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe Met

340 345 350

Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu

355 360 365

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

370 375 380

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

385 390 395 400

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

405 410 415

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

420 425 430

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

435 440 445

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

450 455 460

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

465 470 475 480

Arg

<210> 210

<211> 1443

<212> DNA

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR ICOS CD3z

<400> 210

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatttc tggttaccca taggatgtgc agcctttgtt 960

gtagtctgca ttttgggatg catacttatt tgttggctta caaaaaagaa gtattcatcc 1020

agtgtgcacg accctaacgg tgaatacatg ttcatgagag cagtgaacac agccaaaaaa 1080

tccagactca cagatgtgac cctaagagtg aagttcagca ggagcgcaga cgcccccgcg 1140

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgc 1443

<210> 211

<211> 481

<212> PRT

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR varICOS CD3z

<400> 211

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val

20 25 30

Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr

35 40 45

Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys

50 55 60

Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg

65 70 75 80

Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser

85 90 95

Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr

100 105 110

Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp

115 120 125

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser

195 200 205

Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val

305 310 315 320

Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys

325 330 335

Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Asn Met

340 345 350

Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu

355 360 365

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

370 375 380

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

385 390 395 400

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

405 410 415

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

420 425 430

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

435 440 445

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

450 455 460

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

465 470 475 480

Arg

<210> 212

<211> 1443

<212> DNA

<213> Artificial sequence

<220>

<223> 2F5 human PSMA-CAR varICOS CD3z

<400> 212

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 120

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 180

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 240

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 300

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 360

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 600

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 660

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 720

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 780

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatttc tggttaccca taggatgtgc agcctttgtt 960

gtagtctgca ttttgggatg catacttatt tgttggctta caaaaaagaa gtattcatcc 1020

agtgtgcacg accctaacgg tgaatacatg aacatgagag cagtgaacac agccaaaaaa 1080

tccagactca cagatgtgac cctaagagtg aagttcagca ggagcgcaga cgcccccgcg 1140

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgc 1443

<210> 213

<211> 237

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-4-1BB transform receptor

<400> 213

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

225 230 235

<210> 214

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-4-1BB transform receptor

<400> 214

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g 711

<210> 215

<211> 237

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB transform receptor

<400> 215

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

225 230 235

<210> 216

<211> 711

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB transform receptor

<400> 216

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact g 711

<210> 217

<211> 2223

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CD28-F2A-2F5PSMA-CAR ICOS CD3z

<400> 217

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 960

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 1020

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 1080

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 1140

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 1260

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 1380

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 1440

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 1500

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 1560

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1680

acgagggggc tggacttcgc ctgtgatttc tggttaccca taggatgtgc agcctttgtt 1740

gtagtctgca ttttgggatg catacttatt tgttggctta caaaaaagaa gtattcatcc 1800

agtgtgcacg accctaacgg tgaatacatg ttcatgagag cagtgaacac agccaaaaaa 1860

tccagactca cagatgtgac cctaagagtg aagttcagca ggagcgcaga cgcccccgcg 1920

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1980

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg 2040

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2100

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 2160

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 2220

cgc 2223

<210> 218

<211> 2223

<212> DNA

<213> Artificial sequence

<220>

<223> PD1-CD28-2F5PSMA-CAR varICOS CD3z

<400> 218

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540

ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600

aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660

cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccgtgaaa 720

cagactttga attttgacct tctcaagttg gcgggagacg tggagtccaa cccagggccg 780

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 840

ccggaggtgc agctggtgca gtctggagca gaggtgaaaa agcccgggga gtctctgaag 900

atctcctgta agggttctgg atacagtttt accagcaact ggatcggctg ggtgcgccag 960

atgcccggga aaggcctgga gtggatgggg atcatctatc ctggtgactc tgataccaga 1020

tacagcccgt ccttccaagg ccaggtcacc atctcagccg acaagtccat cagcaccgcc 1080

tacctgcagt ggaacagcct gaaggcctcg gacaccgcca tgtattactg tgcgagacaa 1140

actggtttcc tctggtcctt cgatctctgg ggccgtggca ccctggtcac tgtctcctca 1200

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgccat ccagttgacc 1260

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 1320

agtcaggaca ttagcagtgc tttagcctgg tatcagcaga aaccggggaa agctcctaag 1380

ctcctgatct atgatgcctc cagtttggaa agtggggtcc catcaaggtt cagcggcagt 1440

ggatctggga cagatttcac tctcaccatc agcagcctgc agcctgaaga ttttgcaact 1500

tattactgtc aacagtttaa tagttacccg ctcactttcg gcggagggac caaggtggag 1560

atcaaaatca aaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1680

acgagggggc tggacttcgc ctgtgatttc tggttaccca taggatgtgc agcctttgtt 1740

gtagtctgca ttttgggatg catacttatt tgttggctta caaaaaagaa gtattcatcc 1800

agtgtgcacg accctaacgg tgaatacatg aacatgagag cagtgaacac agccaaaaaa 1860

tccagactca cagatgtgac cctaagagtg aagttcagca ggagcgcaga cgcccccgcg 1920

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1980

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg 2040

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 2100

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 2160

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 2220

cgc 2223

<210> 219

<211> 2205

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-CD28-2F5PSMA-CAR ICOS CD3z

<400> 219

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt gcagctggtg 840

cagtctggag cagaggtgaa aaagcccggg gagtctctga agatctcctg taagggttct 900

ggatacagtt ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg 960

gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc gtccttccaa 1020

ggccaggtca ccatctcagc cgacaagtcc atcagcaccg cctacctgca gtggaacagc 1080

ctgaaggcct cggacaccgc catgtattac tgtgcgagac aaactggttt cctctggtcc 1140

ttcgatctct ggggccgtgg caccctggtc actgtctcct caggtggcgg tggctcgggc 1200

ggtggtgggt cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg 1260

tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga cattagcagt 1320

gctttagcct ggtatcagca gaaaccgggg aaagctccta agctcctgat ctatgatgcc 1380

tccagtttgg aaagtggggt cccatcaagg ttcagcggca gtggatctgg gacagatttc 1440

actctcacca tcagcagcct gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500

aatagttacc cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg 1560

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 1620

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 1680

gcctgtgatt tctggttacc cataggatgt gcagcctttg ttgtagtctg cattttggga 1740

tgcatactta tttgttggct tacaaaaaag aagtattcat ccagtgtgca cgaccctaac 1800

ggtgaataca tgttcatgag agcagtgaac acagccaaaa aatccagact cacagatgtg 1860

accctaagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1920

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1980

cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 2040

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 2100

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 2160

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 2205

<210> 220

<211> 2205

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-CD28-2F5PSMA-CAR varICOS CD3z

<400> 220

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atcaggagta agaggagcag gctcctgcac 600

agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 660

tatgccccac cacgcgactt cgcagcctat cgctccgtga aacagacttt gaattttgac 720

cttctcaagt tggcgggaga cgtggagtcc aacccagggc cgatggcctt accagtgacc 780

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccggaggt gcagctggtg 840

cagtctggag cagaggtgaa aaagcccggg gagtctctga agatctcctg taagggttct 900

ggatacagtt ttaccagcaa ctggatcggc tgggtgcgcc agatgcccgg gaaaggcctg 960

gagtggatgg ggatcatcta tcctggtgac tctgatacca gatacagccc gtccttccaa 1020

ggccaggtca ccatctcagc cgacaagtcc atcagcaccg cctacctgca gtggaacagc 1080

ctgaaggcct cggacaccgc catgtattac tgtgcgagac aaactggttt cctctggtcc 1140

ttcgatctct ggggccgtgg caccctggtc actgtctcct caggtggcgg tggctcgggc 1200

ggtggtgggt cgggtggcgg cggatctgcc atccagttga cccagtctcc atcctccctg 1260

tctgcatctg taggagacag agtcaccatc acttgccggg caagtcagga cattagcagt 1320

gctttagcct ggtatcagca gaaaccgggg aaagctccta agctcctgat ctatgatgcc 1380

tccagtttgg aaagtggggt cccatcaagg ttcagcggca gtggatctgg gacagatttc 1440

actctcacca tcagcagcct gcagcctgaa gattttgcaa cttattactg tcaacagttt 1500

aatagttacc cgctcacttt cggcggaggg accaaggtgg agatcaaaat caaaaccacg 1560

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 1620

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 1680

gcctgtgatt tctggttacc cataggatgt gcagcctttg ttgtagtctg cattttggga 1740

tgcatactta tttgttggct tacaaaaaag aagtattcat ccagtgtgca cgaccctaac 1800

ggtgaataca tgaacatgag agcagtgaac acagccaaaa aatccagact cacagatgtg 1860

accctaagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1920

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1980

cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 2040

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 2100

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 2160

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 2205

<210> 221

<211> 2220

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-41BB-2F5PSMA-CAR ICOS CD3z

<400> 221

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggtgaaacag 720

actttgaatt ttgaccttct caagttggcg ggagacgtgg agtccaaccc agggccgatg 780

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 840

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 900

tcctgtaagg gttctggata cagttttacc agcaactgga tcggctgggt gcgccagatg 960

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 1020

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 1080

ctgcagtgga acagcctgaa ggcctcggac accgccatgt attactgtgc gagacaaact 1140

ggtttcctct ggtccttcga tctctggggc cgtggcaccc tggtcactgt ctcctcaggt 1200

ggcggtggct cgggcggtgg tgggtcgggt ggcggcggat ctgccatcca gttgacccag 1260

tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 1320

caggacatta gcagtgcttt agcctggtat cagcagaaac cggggaaagc tcctaagctc 1380

ctgatctatg atgcctccag tttggaaagt ggggtcccat caaggttcag cggcagtgga 1440

tctgggacag atttcactct caccatcagc agcctgcagc ctgaagattt tgcaacttat 1500

tactgtcaac agtttaatag ttacccgctc actttcggcg gagggaccaa ggtggagatc 1560

aaaatcaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1620

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 1680

agggggctgg acttcgcctg tgatttctgg ttacccatag gatgtgcagc ctttgttgta 1740

gtctgcattt tgggatgcat acttatttgt tggcttacaa aaaagaagta ttcatccagt 1800

gtgcacgacc ctaacggtga atacatgttc atgagagcag tgaacacagc caaaaaatcc 1860

agactcacag atgtgaccct aagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1920

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1980

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 2040

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 2100

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 2160

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 2220

<210> 222

<211> 2220

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-41BB-2F5PSMA-CAR varICOS CD3z

<400> 222

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggtgaaacag 720

actttgaatt ttgaccttct caagttggcg ggagacgtgg agtccaaccc agggccgatg 780

gccttaccag tgaccgcctt gctcctgccg ctggccttgc tgctccacgc cgccaggccg 840

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 900

tcctgtaagg gttctggata cagttttacc agcaactgga tcggctgggt gcgccagatg 960

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 1020

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 1080

ctgcagtgga acagcctgaa ggcctcggac accgccatgt attactgtgc gagacaaact 1140

ggtttcctct ggtccttcga tctctggggc cgtggcaccc tggtcactgt ctcctcaggt 1200

ggcggtggct cgggcggtgg tgggtcgggt ggcggcggat ctgccatcca gttgacccag 1260

tctccatcct ccctgtctgc atctgtagga gacagagtca ccatcacttg ccgggcaagt 1320

caggacatta gcagtgcttt agcctggtat cagcagaaac cggggaaagc tcctaagctc 1380

ctgatctatg atgcctccag tttggaaagt ggggtcccat caaggttcag cggcagtgga 1440

tctgggacag atttcactct caccatcagc agcctgcagc ctgaagattt tgcaacttat 1500

tactgtcaac agtttaatag ttacccgctc actttcggcg gagggaccaa ggtggagatc 1560

aaaatcaaaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 1620

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 1680

agggggctgg acttcgcctg tgatttctgg ttacccatag gatgtgcagc ctttgttgta 1740

gtctgcattt tgggatgcat acttatttgt tggcttacaa aaaagaagta ttcatccagt 1800

gtgcacgacc ctaacggtga atacatgaac atgagagcag tgaacacagc caaaaaatcc 1860

agactcacag atgtgaccct aagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1920

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1980

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 2040

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 2100

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 2160

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 2220

<210> 223

<211> 2313

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-2F5PSMA-CAR ICOS CD3z

<400> 223

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca 960

gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg atacagtttt 1020

accagcaact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 1140

atctcagccg acaagtccat cagcaccgcc tacctgcagt ggaacagcct gaaggcctcg 1200

gacaccgcca tgtattactg tgcgagacaa actggtttcc tctggtcctt cgatctctgg 1260

ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg tggtgggtcg 1320

ggtggcggcg gatctgccat ccagttgacc cagtctccat cctccctgtc tgcatctgta 1380

ggagacagag tcaccatcac ttgccgggca agtcaggaca ttagcagtgc tttagcctgg 1440

tatcagcaga aaccggggaa agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500

agtggggtcc catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc 1560

agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa tagttacccg 1620

ctcactttcg gcggagggac caaggtggag atcaaaatca aaaccacgac gccagcgccg 1680

cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1740

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatttc 1800

tggttaccca taggatgtgc agcctttgtt gtagtctgca ttttgggatg catacttatt 1860

tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg tgaatacatg 1920

ttcatgagag cagtgaacac agccaaaaaa tccagactca cagatgtgac cctaagagtg 1980

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 2040

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 2100

cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 2160

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 2220

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 2280

gacgcccttc acatgcaggc cctgccccct cgc 2313

<210> 224

<211> 2313

<212> DNA

<213> Artificial sequence

<220>

<223> TIM3-CD28-2F5PSMA-CAR varICOS CD3z

<400> 224

atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg 60

tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac 120

accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg 180

tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc 240

agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg 300

actctagcag acagtgggat ctactgctgc cgaatccaaa tcccaggcat aatgaatgat 360

gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg 420

cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca 480

gagacacaga cactggggag cctccctgac ataaatctaa cacaaatatc cacattggcc 540

aatgagttac gggactctag gttggccaat gacttacggg actccggagc aaccatcaga 600

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttact agtaacagtg 660

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 720

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 780

cgcgacttcg cagcctatcg ctccgtgaaa cagactttga attttgacct tctcaagttg 840

gcgggagacg tggagtccaa cccagggccg atggccttac cagtgaccgc cttgctcctg 900

ccgctggcct tgctgctcca cgccgccagg ccggaggtgc agctggtgca gtctggagca 960

gaggtgaaaa agcccgggga gtctctgaag atctcctgta agggttctgg atacagtttt 1020

accagcaact ggatcggctg ggtgcgccag atgcccggga aaggcctgga gtggatgggg 1080

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg ccaggtcacc 1140

atctcagccg acaagtccat cagcaccgcc tacctgcagt ggaacagcct gaaggcctcg 1200

gacaccgcca tgtattactg tgcgagacaa actggtttcc tctggtcctt cgatctctgg 1260

ggccgtggca ccctggtcac tgtctcctca ggtggcggtg gctcgggcgg tggtgggtcg 1320

ggtggcggcg gatctgccat ccagttgacc cagtctccat cctccctgtc tgcatctgta 1380

ggagacagag tcaccatcac ttgccgggca agtcaggaca ttagcagtgc tttagcctgg 1440

tatcagcaga aaccggggaa agctcctaag ctcctgatct atgatgcctc cagtttggaa 1500

agtggggtcc catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc 1560

agcagcctgc agcctgaaga ttttgcaact tattactgtc aacagtttaa tagttacccg 1620

ctcactttcg gcggagggac caaggtggag atcaaaatca aaaccacgac gccagcgccg 1680

cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg 1740

tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatttc 1800

tggttaccca taggatgtgc agcctttgtt gtagtctgca ttttgggatg catacttatt 1860

tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg tgaatacatg 1920

aacatgagag cagtgaacac agccaaaaaa tccagactca cagatgtgac cctaagagtg 1980

aagttcagca ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac 2040

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 2100

cctgagatgg ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa 2160

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 2220

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 2280

gacgcccttc acatgcaggc cctgccccct cgc 2313

<210> 225

<211> 3090

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR ICOS CD3z

<400> 225

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggtgaagcag 720

acgttgaact tcgatttgct caaacttgcc ggtgacgtgg aatccaatcc ggggccgatg 780

ttttcacatc ttccctttga ctgtgtcctg ctgctgctgc tgctactact tacaaggtcc 840

tcagaagtgg aatacagagc ggaggtcggt cagaatgcct atctgccctg cttctacacc 900

ccagccgccc cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt 960

gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg gacatccaga 1020

tactggctaa atggggattt ccgcaaagga gatgtgtccc tgaccataga gaatgtgact 1080

ctagcagaca gtgggatcta ctgctgccga atccaaatcc caggcataat gaatgatgaa 1140

aaatttaacc tgaagttggt catcaaacca gccaaggtca cccctgcacc gactcggcag 1200

agagacttca ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag 1260

acacagacac tggggagcct ccctgacata aatctaacac aaatatccac attggccaat 1320

gagttacggg actctaggtt ggccaatgac ttacgggact ccggagcaac catcagattt 1380

tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttactagt aacagtggcc 1440

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1500

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1560

gacttcgcag cctatcgctc cgtgaaacag actttgaatt ttgaccttct caagttggcg 1620

ggagacgtgg agtccaaccc agggccgatg gccttaccag tgaccgcctt gctcctgccg 1680

ctggccttgc tgctccacgc cgccaggccg gaggtgcagc tggtgcagtc tggagcagag 1740

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagttttacc 1800

agcaactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 1860

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 1920

tcagccgaca agtccatcag caccgcctac ctgcagtgga acagcctgaa ggcctcggac 1980

accgccatgt attactgtgc gagacaaact ggtttcctct ggtccttcga tctctggggc 2040

cgtggcaccc tggtcactgt ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt 2100

ggcggcggat ctgccatcca gttgacccag tctccatcct ccctgtctgc atctgtagga 2160

gacagagtca ccatcacttg ccgggcaagt caggacatta gcagtgcttt agcctggtat 2220

cagcagaaac cggggaaagc tcctaagctc ctgatctatg atgcctccag tttggaaagt 2280

ggggtcccat caaggttcag cggcagtgga tctgggacag atttcactct caccatcagc 2340

agcctgcagc ctgaagattt tgcaacttat tactgtcaac agtttaatag ttacccgctc 2400

actttcggcg gagggaccaa ggtggagatc aaaatcaaaa ccacgacgcc agcgccgcga 2460

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 2520

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatttctgg 2580

ttacccatag gatgtgcagc ctttgttgta gtctgcattt tgggatgcat acttatttgt 2640

tggcttacaa aaaagaagta ttcatccagt gtgcacgacc ctaacggtga atacatgttc 2700

atgagagcag tgaacacagc caaaaaatcc agactcacag atgtgaccct aagagtgaag 2760

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 2820

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 2880

gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 2940

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 3000

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 3060

gcccttcaca tgcaggccct gccccctcgc 3090

<210> 226

<211> 3090

<212> DNA

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR varICOS CD3z

<400> 226

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aagctgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtt atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaaaaacg gggcagaaag 600

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 660

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggtgaagcag 720

acgttgaact tcgatttgct caaacttgcc ggtgacgtgg aatccaatcc ggggccgatg 780

ttttcacatc ttccctttga ctgtgtcctg ctgctgctgc tgctactact tacaaggtcc 840

tcagaagtgg aatacagagc ggaggtcggt cagaatgcct atctgccctg cttctacacc 900

ccagccgccc cagggaacct cgtgcccgtc tgctggggca aaggagcctg tcctgtgttt 960

gaatgtggca acgtggtgct caggactgat gaaagggatg tgaattattg gacatccaga 1020

tactggctaa atggggattt ccgcaaagga gatgtgtccc tgaccataga gaatgtgact 1080

ctagcagaca gtgggatcta ctgctgccga atccaaatcc caggcataat gaatgatgaa 1140

aaatttaacc tgaagttggt catcaaacca gccaaggtca cccctgcacc gactcggcag 1200

agagacttca ctgcagcctt tccaaggatg cttaccacca ggggacatgg cccagcagag 1260

acacagacac tggggagcct ccctgacata aatctaacac aaatatccac attggccaat 1320

gagttacggg actctaggtt ggccaatgac ttacgggact ccggagcaac catcagattt 1380

tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttactagt aacagtggcc 1440

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1500

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1560

gacttcgcag cctatcgctc cgtgaaacag actttgaatt ttgaccttct caagttggcg 1620

ggagacgtgg agtccaaccc agggccgatg gccttaccag tgaccgcctt gctcctgccg 1680

ctggccttgc tgctccacgc cgccaggccg gaggtgcagc tggtgcagtc tggagcagag 1740

gtgaaaaagc ccggggagtc tctgaagatc tcctgtaagg gttctggata cagttttacc 1800

agcaactgga tcggctgggt gcgccagatg cccgggaaag gcctggagtg gatggggatc 1860

atctatcctg gtgactctga taccagatac agcccgtcct tccaaggcca ggtcaccatc 1920

tcagccgaca agtccatcag caccgcctac ctgcagtgga acagcctgaa ggcctcggac 1980

accgccatgt attactgtgc gagacaaact ggtttcctct ggtccttcga tctctggggc 2040

cgtggcaccc tggtcactgt ctcctcaggt ggcggtggct cgggcggtgg tgggtcgggt 2100

ggcggcggat ctgccatcca gttgacccag tctccatcct ccctgtctgc atctgtagga 2160

gacagagtca ccatcacttg ccgggcaagt caggacatta gcagtgcttt agcctggtat 2220

cagcagaaac cggggaaagc tcctaagctc ctgatctatg atgcctccag tttggaaagt 2280

ggggtcccat caaggttcag cggcagtgga tctgggacag atttcactct caccatcagc 2340

agcctgcagc ctgaagattt tgcaacttat tactgtcaac agtttaatag ttacccgctc 2400

actttcggcg gagggaccaa ggtggagatc aaaatcaaaa ccacgacgcc agcgccgcga 2460

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 2520

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatttctgg 2580

ttacccatag gatgtgcagc ctttgttgta gtctgcattt tgggatgcat acttatttgt 2640

tggcttacaa aaaagaagta ttcatccagt gtgcacgacc ctaacggtga atacatgaac 2700

atgagagcag tgaacacagc caaaaaatcc agactcacag atgtgaccct aagagtgaag 2760

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 2820

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 2880

gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 2940

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 3000

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 3060

gcccttcaca tgcaggccct gccccctcgc 3090

<210> 227

<211> 741

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-CD28-2F5PSMA-CAR ICOS CD3z

<400> 227

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Phe Trp Val Leu Val Val

165 170 175

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

180 185 190

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

195 200 205

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

210 215 220

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys

225 230 235 240

Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

245 250 255

Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu

260 265 270

Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser

275 280 285

Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys

290 295 300

Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln

305 310 315 320

Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp

325 330 335

Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser

340 345 350

Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys

355 360 365

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu

370 375 380

Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

405 410 415

Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

420 425 430

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu

435 440 445

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

450 455 460

Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

465 470 475 480

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

485 490 495

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr

500 505 510

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro

515 520 525

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

530 535 540

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

545 550 555 560

Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys

565 570 575

Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp

580 585 590

Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu

595 600 605

Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr

610 615 620

Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

625 630 635 640

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

645 650 655

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

660 665 670

Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

675 680 685

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

690 695 700

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

705 710 715 720

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

725 730 735

Ala Leu Pro Pro Arg

740

<210> 228

<211> 735

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-CD28-2F5PSMA-CAR varICOS CD3z

<400> 228

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg

180 185 190

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

195 200 205

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

210 215 220

Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr Leu Asn Phe Asp

225 230 235 240

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala

245 250 255

Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala

260 265 270

Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

275 280 285

Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

290 295 300

Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

305 310 315 320

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

325 330 335

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

340 345 350

Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met

355 360 365

Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp Leu Trp

370 375 380

Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser

405 410 415

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

420 425 430

Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys

435 440 445

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu

450 455 460

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

465 470 475 480

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

485 490 495

Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys

500 505 510

Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

515 520 525

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

530 535 540

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

545 550 555 560

Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val

565 570 575

Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr

580 585 590

Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala

595 600 605

Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Arg Val

610 615 620

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

625 630 635 640

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

645 650 655

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

660 665 670

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

675 680 685

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

690 695 700

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

705 710 715 720

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

725 730 735

<210> 229

<211> 740

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-41BB-2F5PSMA-CAR ICOS CD3z

<400> 229

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val Lys Gln

225 230 235 240

Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

245 250 255

Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

260 265 270

Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly

275 280 285

Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly

290 295 300

Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met

305 310 315 320

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser

325 330 335

Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala

340 345 350

Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala

355 360 365

Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp

370 375 380

Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile

405 410 415

Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

420 425 430

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala

435 440 445

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp

450 455 460

Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

485 490 495

Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe

500 505 510

Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala

515 520 525

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

530 535 540

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

545 550 555 560

Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala

565 570 575

Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu

580 585 590

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

595 600 605

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

610 615 620

Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

625 630 635 640

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

645 650 655

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

660 665 670

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

675 680 685

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

690 695 700

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

705 710 715 720

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

725 730 735

Leu Pro Pro Arg

740

<210> 230

<211> 771

<212> PRT

<213> Artificial sequence

<220>

<223> TIM3-CD28-2F5PSMA-CAR ICOS CD3z

<400> 230

Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln

20 25 30

Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu

35 40 45

Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly

50 55 60

Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser

65 70 75 80

Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr

85 90 95

Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile

100 105 110

Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val

115 120 125

Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe

130 135 140

Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala

145 150 155 160

Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile

165 170 175

Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu

180 185 190

Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu Val Val Val Gly

195 200 205

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

210 215 220

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

225 230 235 240

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

245 250 255

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr

260 265 270

Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro

275 280 285

Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu

290 295 300

Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala

305 310 315 320

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

325 330 335

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

340 345 350

Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp

355 360 365

Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

370 375 380

Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser

385 390 395 400

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser

405 410 415

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly

420 425 430

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln

435 440 445

Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

450 455 460

Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp

465 470 475 480

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala

485 490 495

Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

500 505 510

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

515 520 525

Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly

530 535 540

Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro

545 550 555 560

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

565 570 575

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

580 585 590

Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala

595 600 605

Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr

610 615 620

Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met

625 630 635 640

Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val

645 650 655

Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

660 665 670

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

675 680 685

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

690 695 700

Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

705 710 715 720

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

725 730 735

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

740 745 750

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

755 760 765

Pro Pro Arg

770

<210> 231

<211> 1030

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR ICOS CD3z

<400> 231

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val Lys Gln

225 230 235 240

Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

245 250 255

Pro Gly Pro Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu

260 265 270

Leu Leu Leu Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu

275 280 285

Val Gly Gln Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro

290 295 300

Gly Asn Leu Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe

305 310 315 320

Glu Cys Gly Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr

325 330 335

Trp Thr Ser Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val

340 345 350

Ser Leu Thr Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys

355 360 365

Cys Arg Ile Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu

370 375 380

Lys Leu Val Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln

385 390 395 400

Arg Asp Phe Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His

405 410 415

Gly Pro Ala Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu

420 425 430

Thr Gln Ile Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala

435 440 445

Asn Asp Leu Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu Val

450 455 460

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

465 470 475 480

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

485 490 495

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

500 505 510

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val

515 520 525

Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu

530 535 540

Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro

545 550 555 560

Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln

565 570 575

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

580 585 590

Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg

595 600 605

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly

610 615 620

Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

625 630 635 640

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu

645 650 655

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe

660 665 670

Leu Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser

675 680 685

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

690 695 700

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

705 710 715 720

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala

725 730 735

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

740 745 750

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

755 760 765

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

770 775 780

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

785 790 795 800

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr

805 810 815

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

820 825 830

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

835 840 845

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly

850 855 860

Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys

865 870 875 880

Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly

885 890 895

Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu

900 905 910

Thr Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

915 920 925

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

930 935 940

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

945 950 955 960

Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu

965 970 975

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

980 985 990

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

995 1000 1005

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

1010 1015 1020

Met Gln Ala Leu Pro Pro Arg

1025 1030

<210> 232

<211> 741

<212> PRT

<213> Artificial sequence

<220>

<223> PD1-CD28-2F5PSMA-CAR varICOS CD3z

<400> 232

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Phe Trp Val Leu Val Val

165 170 175

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

180 185 190

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

195 200 205

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

210 215 220

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys

225 230 235 240

Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

245 250 255

Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu

260 265 270

Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser

275 280 285

Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys

290 295 300

Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln

305 310 315 320

Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp

325 330 335

Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser

340 345 350

Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys

355 360 365

Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu

370 375 380

Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

405 410 415

Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp

420 425 430

Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu

435 440 445

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

450 455 460

Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

465 470 475 480

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

485 490 495

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr

500 505 510

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro

515 520 525

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

530 535 540

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

545 550 555 560

Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys

565 570 575

Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp

580 585 590

Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu

595 600 605

Tyr Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr

610 615 620

Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

625 630 635 640

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

645 650 655

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

660 665 670

Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

675 680 685

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

690 695 700

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

705 710 715 720

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

725 730 735

Ala Leu Pro Pro Arg

740

<210> 233

<211> 735

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-CD28-2F5PSMA-CAR ICOS CD3z

<400> 233

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg

180 185 190

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

195 200 205

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

210 215 220

Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr Leu Asn Phe Asp

225 230 235 240

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala

245 250 255

Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala

260 265 270

Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

275 280 285

Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

290 295 300

Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

305 310 315 320

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

325 330 335

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

340 345 350

Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met

355 360 365

Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser Phe Asp Leu Trp

370 375 380

Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln Leu Thr Gln Ser

405 410 415

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

420 425 430

Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp Tyr Gln Gln Lys

435 440 445

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu

450 455 460

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

465 470 475 480

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

485 490 495

Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys

500 505 510

Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

515 520 525

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

530 535 540

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

545 550 555 560

Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val

565 570 575

Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr

580 585 590

Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Asn Met Arg Ala

595 600 605

Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu Arg Val

610 615 620

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

625 630 635 640

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

645 650 655

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln

660 665 670

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

675 680 685

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

690 695 700

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

705 710 715 720

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

725 730 735

<210> 234

<211> 740

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-41BB-2F5PSMA-CAR varICOS CD3z

<400> 234

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val Lys Gln

225 230 235 240

Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

245 250 255

Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

260 265 270

Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly

275 280 285

Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly

290 295 300

Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met

305 310 315 320

Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser

325 330 335

Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala

340 345 350

Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala

355 360 365

Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp

370 375 380

Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile

405 410 415

Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg

420 425 430

Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala

435 440 445

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp

450 455 460

Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

485 490 495

Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe

500 505 510

Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala

515 520 525

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

530 535 540

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

545 550 555 560

Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala

565 570 575

Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu

580 585 590

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

595 600 605

Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

610 615 620

Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

625 630 635 640

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

645 650 655

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

660 665 670

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

675 680 685

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

690 695 700

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

705 710 715 720

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

725 730 735

Leu Pro Pro Arg

740

<210> 235

<211> 771

<212> PRT

<213> Artificial sequence

<220>

<223> TIM3-CD28-2F5PSMA-CAR varICOS CD3z

<400> 235

Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu Leu Leu Leu

1 5 10 15

Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln

20 25 30

Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu

35 40 45

Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly

50 55 60

Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser

65 70 75 80

Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr

85 90 95

Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile

100 105 110

Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val

115 120 125

Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe

130 135 140

Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala

145 150 155 160

Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile

165 170 175

Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu

180 185 190

Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu Val Val Val Gly

195 200 205

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

210 215 220

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

225 230 235 240

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

245 250 255

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Gln Thr

260 265 270

Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro

275 280 285

Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu

290 295 300

Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln Ser Gly Ala

305 310 315 320

Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser

325 330 335

Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg Gln Met Pro

340 345 350

Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp

355 360 365

Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp

370 375 380

Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser

385 390 395 400

Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe Leu Trp Ser

405 410 415

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly

420 425 430

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ile Gln

435 440 445

Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val

450 455 460

Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala Leu Ala Trp

465 470 475 480

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala

485 490 495

Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

500 505 510

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

515 520 525

Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu Thr Phe Gly

530 535 540

Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr Pro Ala Pro

545 550 555 560

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

565 570 575

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

580 585 590

Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala Ala

595 600 605

Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu Thr

610 615 620

Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met

625 630 635 640

Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val

645 650 655

Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

660 665 670

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

675 680 685

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

690 695 700

Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

705 710 715 720

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

725 730 735

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

740 745 750

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

755 760 765

Pro Pro Arg

770

<210> 236

<211> 1030

<212> PRT

<213> Artificial sequence

<220>

<223> PD1A132L-4-1BB-TIM3-CD28-2F5PSMA-CAR varICOS CD3z

<400> 236

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Leu Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Lys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

195 200 205

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

210 215 220

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Val Lys Gln

225 230 235 240

Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

245 250 255

Pro Gly Pro Met Phe Ser His Leu Pro Phe Asp Cys Val Leu Leu Leu

260 265 270

Leu Leu Leu Leu Leu Thr Arg Ser Ser Glu Val Glu Tyr Arg Ala Glu

275 280 285

Val Gly Gln Asn Ala Tyr Leu Pro Cys Phe Tyr Thr Pro Ala Ala Pro

290 295 300

Gly Asn Leu Val Pro Val Cys Trp Gly Lys Gly Ala Cys Pro Val Phe

305 310 315 320

Glu Cys Gly Asn Val Val Leu Arg Thr Asp Glu Arg Asp Val Asn Tyr

325 330 335

Trp Thr Ser Arg Tyr Trp Leu Asn Gly Asp Phe Arg Lys Gly Asp Val

340 345 350

Ser Leu Thr Ile Glu Asn Val Thr Leu Ala Asp Ser Gly Ile Tyr Cys

355 360 365

Cys Arg Ile Gln Ile Pro Gly Ile Met Asn Asp Glu Lys Phe Asn Leu

370 375 380

Lys Leu Val Ile Lys Pro Ala Lys Val Thr Pro Ala Pro Thr Arg Gln

385 390 395 400

Arg Asp Phe Thr Ala Ala Phe Pro Arg Met Leu Thr Thr Arg Gly His

405 410 415

Gly Pro Ala Glu Thr Gln Thr Leu Gly Ser Leu Pro Asp Ile Asn Leu

420 425 430

Thr Gln Ile Ser Thr Leu Ala Asn Glu Leu Arg Asp Ser Arg Leu Ala

435 440 445

Asn Asp Leu Arg Asp Ser Gly Ala Thr Ile Arg Phe Trp Val Leu Val

450 455 460

Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala

465 470 475 480

Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser

485 490 495

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

500 505 510

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val

515 520 525

Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu

530 535 540

Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro

545 550 555 560

Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Gln Leu Val Gln

565 570 575

Ser Gly Ala Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys

580 585 590

Lys Gly Ser Gly Tyr Ser Phe Thr Ser Asn Trp Ile Gly Trp Val Arg

595 600 605

Gln Met Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly

610 615 620

Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile

625 630 635 640

Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Asn Ser Leu

645 650 655

Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Thr Gly Phe

660 665 670

Leu Trp Ser Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser

675 680 685

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

690 695 700

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

705 710 715 720

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala

725 730 735

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

740 745 750

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

755 760 765

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

770 775 780

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu

785 790 795 800

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ile Lys Thr Thr Thr

805 810 815

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

820 825 830

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

835 840 845

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly

850 855 860

Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys

865 870 875 880

Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly

885 890 895

Glu Tyr Met Asn Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu

900 905 910

Thr Asp Val Thr Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

915 920 925

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

930 935 940

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

945 950 955 960

Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu

965 970 975

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

980 985 990

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

995 1000 1005

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

1010 1015 1020

Met Gln Ala Leu Pro Pro Arg

1025 1030

Claims (95)

1. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for a prostate-specific membrane antigen on a target cell, wherein the CAR comprises a PSMA binding domain; and

Dominant negative receptors and/or switch receptors.

2. The modified cell of claim 1, wherein the PSMA-binding domain is a murine PSMA-binding domain.

3. The modified cell of claim 1, wherein the PSMA-binding domain is a human PSMA-binding domain.

4. The modified cell of any preceding claim, wherein the PSMA-binding domain is selected from an antibody, Fab, or scFv.

5. The modified cell of any preceding claim, wherein the scFv comprises the amino acid sequence recited in any one of SEQ ID NOs 13 or 14 when the scFv is murine, and wherein the scFv comprises the amino acid sequence recited in any one of SEQ ID NOs 26, 38, 50, or 62 when the scFv is human.

6. The modified cell of any preceding claim, wherein the CAR comprises a transmembrane domain and an intracellular domain.

7. The modified cell of claim 6, wherein the transmembrane domain comprises a transmembrane region derived from CD 8.

8. The modified cell of claim 7, wherein the transmembrane region derived from CD8 comprises the amino acid sequence set forth in SEQ ID NO 88.

9. The modified cell of any one of claims 6-8, wherein the transmembrane domain further comprises a hinge region derived from CD 8.

10. The modified cell of claim 9, wherein the hinge region derived from CD8 comprises the amino acid sequence set forth in SEQ ID No. 86.

11. The modified cell of any one of claims 6-10, wherein the endodomain comprises a 4-1BB signaling domain and a CD3 zeta signaling domain.

12. The modified cell of any one of claims 6-10, wherein the endodomain comprises an ICOS signaling domain and a CD3 zeta signaling domain.

13. The modified cell of any one of claims 6-10, wherein the endodomain comprises a variant ICOS signaling domain and a CD3 zeta signaling domain.

14. The modified cell of claim 11, wherein the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID No. 92.

15. The modified cell of claim 12, wherein the ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID No. 203.

16. The modified cell of claim 13, wherein the variant ICOS signaling domain comprises the amino acid sequence set forth in SEQ ID No. 95.

17. The modified cell of any one of claims 11-13, wherein said CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID No. 97 or 100.

18. The modified cell of any preceding claim, wherein the dominant negative receptor is a truncated variant of a wild-type protein associated with a negative signal.

19. The modified cell of claim 17, wherein the truncated variant of the wild-type protein that is associated with a negative signal comprises the amino acid sequence set forth in SEQ ID No. 115.

20. The modified cell of any one of claims 1-16, wherein the switch receptor comprises:

a first domain, wherein the first domain is derived from a first polypeptide that is associated with a negative signal; and

a second domain, wherein the second domain is derived from a second polypeptide that is associated with a positive signal.

21. The modified cell of claim 19, wherein the first domain comprises at least a portion of an extracellular domain of the first polypeptide that is associated with a negative signal, and wherein the second domain comprises at least a portion of an intracellular domain of the second polypeptide that is associated with a positive signal.

22. The modified cell of any preceding claim, wherein the switch receptor further comprises a switch receptor transmembrane domain.

23. The modified cell of claim 21, wherein the switch receptor transmembrane domain comprises:

a transmembrane domain of the first polypeptide associated with a negative signal; or

A transmembrane domain of the second polypeptide associated with a positive signal.

24. The modified cell of any one of claims 19-22, wherein the first polypeptide associated with a negative signal is selected from CTLA4, PD-1, BTLA, TIM-3, and TGF β R.

25. The modified cell of any one of claims 19-23, wherein the second polypeptide associated with positive signaling is selected from the group consisting of CD28, ICOS, 4-1BB, and IL-12R.

26. The modified cell of any one of claims 19-22, wherein the switch receptor comprises:

a first domain comprising at least a portion of the extracellular domain of PD 1;

a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of CD 28; and

a second domain comprising at least a portion of the intracellular domain of CD 28.

27. The modified cell of claim 25, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 117.

28. The modified cell of any one of claims 19-22, wherein the switch receptor comprises:

a first domain comprising at least a portion of the extracellular domain of PD 1;

a switch receptor transmembrane domain comprising at least a portion of the transmembrane domain of PD 1; and

a second domain comprising at least a portion of the intracellular domain of CD 28.

29. The modified cell of claim 27, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 119.

30. The modified cell of claim 27, wherein the first domain comprises at least a portion of the extracellular domain of PD1 comprising a substitution of alanine (a) to leucine (L) at amino acid position 132.

31. The modified cell of claim 29, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 121.

32. The modified cell of any one of claims 19-22, wherein the switch receptor comprises

A first domain comprising at least a portion of the extracellular domain of PD1, comprising a substitution of alanine (a) to leucine (L) at amino acid position 132; and

a second domain comprising at least a portion of the intracellular domain of CD 28.

33. The modified cell of claim 31, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 121.

34. The modified cell of any one of claims 19-21, wherein the switch receptor comprises:

a first domain comprising at least a portion of the extracellular domain of PD1, comprising a substitution of alanine (a) to leucine (L) at amino acid position 132; and

a second domain comprising at least a portion of the intracellular domain of 4-1 BB.

35. The modified cell of claim 33, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 215.

36. The modified cell of any one of claims 19-21, wherein the switch receptor comprises:

a first domain comprising at least a portion of an extracellular domain of TIM-3; and

a second domain comprising at least a portion of the intracellular domain of CD 28.

37. The modified cell of claim 35, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 127.

38. The modified cell of any one of claims 19-21, wherein the switch receptor comprises:

A first domain comprising at least a portion of the extracellular domain of a TGF β R; and

a second domain comprising at least part of the intracellular domain of IL12R β 1.

39. The modified cell of claim 37, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID No. 123.

40. The modified cell of any one of claims 19-21, wherein the switch receptor comprises:

a first domain comprising at least a portion of the extracellular domain of a TGF β R; and

a second domain comprising at least part of the intracellular domain of IL12R β 2.

41. The modified cell of claim 39, wherein the switch receptor comprises the amino acid sequence set forth in SEQ ID NO 125.

42. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

43. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO 213 or 215.

44. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO 117 or 119.

45. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

A transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 121.

46. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 127.

47. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 123.

48. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising an amino acid sequence recited in any of SEQ ID NOs 13, 14, 16, 38, 50, or 62; and

A transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 125.

49. A modified immune cell or precursor cell thereof comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13; and

a transducible receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

50. The modified cell of any preceding claim, wherein the modified cell further comprises a bispecific antibody.

51. The modified cell of claim 49, wherein the bispecific antibody comprises a first antigen-binding domain and a second antigen-binding domain.

52. The modified cell of claim 50, wherein the first antigen binding domain binds a negative signal selected from CTLA4, PD-1, BTLA, TIM-3, and TGF β R.

53. The modified cell of claim 50 or 51, wherein the second antigen-binding domain binds to a costimulatory molecule.

54. The modified cell of claim 52, wherein the co-stimulatory molecule is CD 28.

55. A modified cell according to any preceding claim, wherein the modified cell is a modified T cell.

56. The modified T cell of claim 54, wherein the modified T cell is an autologous cell.

57. The modified cell of any one of claims 1-55, wherein the modified cell is a Cytotoxic T Lymphocyte (CTL).

58. The modified cell of any one of claims 1-53, wherein the modified cell is a Natural Killer (NK) cell.

59. The modified cell of any one of claims 1-53, wherein the modified cell is a hematopoietic stem cell or a hematopoietic progenitor cell.

60. A modified cell according to any preceding claim, wherein the modified cell is an autologous cell.

61. A modified cell according to any preceding claim, wherein the modified cell is derived from a human.

62. The modified T cell of claim 54 or 55, wherein the modified T cell is derived from a human.

63. An isolated nucleic acid comprising:

a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain; and

A second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor.

64. The isolated nucleic acid of claim 62, wherein the first nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 106, 108, 110, 112, 114, 210, 212.

65. The isolated nucleic acid of claim 62 or 63, wherein the second nucleic acid sequence comprises the nucleic acid sequence set forth in any one of SEQ ID NOs 116, 118, 120, 122, 124, 126, 128, 214, or 216.

66. The isolated nucleic acid of any one of claims 62-64, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker.

67. The isolated nucleic acid of claim 65, wherein the linker comprises a nucleic acid sequence encoding an Internal Ribosome Entry Site (IRES).

68. The isolated nucleic acid of claim 65, wherein the linker comprises a nucleic acid sequence encoding a self-cleaving peptide.

69. The isolated nucleic acid of claim 67, wherein the self-cleaving peptide is a 2A peptide.

70. The isolated nucleic acid of claim 68, wherein the 2A peptide is selected from the group consisting of porcine teschovirus-12A (P2A), Gliocladium intybus beta-tetrahexvirus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot and mouth disease virus 2A (F2A).

71. The isolated nucleic acid of claim 68, wherein the 2A peptide is T2A.

72. The isolated nucleic acid of claim 68, wherein the 2A peptide is F2A.

73. The isolated nucleic acid of any one of claims 62-71, wherein the isolated nucleic acid comprises, from 5 'to 3', the first nucleic acid sequence, the linker, and the second nucleic acid sequence.

74. The isolated nucleic acid of any one of claims 62-71, wherein the isolated nucleic acid comprises, from 5 'to 3', the second nucleic acid sequence, the linker, and the first nucleic acid sequence.

75. An isolated nucleic acid comprising:

a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the nucleic acid sequence recited in any of SEQ ID NOs 180, 15, 27, 39, 51, or 63; and

a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor comprising the nucleic acid sequence set forth in any one of SEQ ID NOs 116, 118, 120, 122, 124, 126, 128, 214, or 216.

76. An isolated nucleic acid comprising:

a first nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the nucleic acid sequence set forth in SEQ ID NO: 180; and

a second nucleic acid sequence encoding a dominant negative receptor and/or a switch receptor comprising the nucleic acid sequence set forth in SEQ ID NO: 116.

77. The isolated nucleic acid of claim 75, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding T2A.

78. The isolated nucleic acid of claim 75, wherein the first nucleic acid sequence and the second nucleic acid sequence are separated by a linker comprising a nucleic acid sequence encoding F2A.

79. An isolated nucleic acid comprising the nucleic acid sequence recited in any one of SEQ ID NO 152-168, 210, 212, and 217-226.

80. An isolated nucleic acid comprising the nucleic acid sequence encoding the bispecific antibody recited in any one of SEQ ID NOs 130, 132, 134, 136, or 138.

81. An expression construct comprising the isolated nucleic acid of any one of claims 62-79.

82. The expression construct according to claim 80, wherein said expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

83. The expression construct of claim 81, wherein said expression construct is a lentiviral vector.

84. The expression construct of claim 82, wherein the lentiviral vector further comprises an EF-1 a promoter.

85. The expression construct of claim 82 or 83, wherein the lentiviral vector further comprises a Rev Response Element (RRE).

86. The expression construct of any one of claims 81-84, wherein the lentiviral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

87. The expression construct of any one of claims 81-84 wherein the lentiviral vector further comprises a cPPT sequence.

88. The expression construct of claim 82, wherein the lentiviral vector further comprises an EF-1 a promoter, a Rev Response Element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

89. The expression construct according to any one of claims 81-87, wherein the lentiviral vector is a self-inactivating lentiviral vector.

90. A method of producing the modified immune cell of any one of claims 1-61 or a precursor cell thereof, comprising introducing one or more of the nucleic acid of any one of claims 62-79 or the expression construct of any one of claims 80-88 into the immune cell.

91. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the modified immune cell of any one of claims 1-61.

92. The method of claim 90, further comprising administering to the subject a lymphodepleting chemotherapy.

93. The method of claim 91, wherein the lymphodepleting chemotherapy comprises administering to the subject a therapeutically effective amount of cyclophosphamide and/or fludarabine.

94. A method of treating prostate cancer in a subject in need thereof, the method comprising:

administering to the subject a lymphodepleting chemotherapy comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and

administering to the subject a modified T cell comprising:

A Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13; and

a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

95. A method of treating metastatic castration-resistant prostate cancer in a subject in need thereof, the method comprising:

administering to the subject a lymphodepleting chemotherapy comprising administering to the subject a therapeutically effective amount of cyclophosphamide; and

administering to the subject a modified T cell comprising:

a Chimeric Antigen Receptor (CAR) having affinity for Prostate Specific Membrane Antigen (PSMA) on a target cell, wherein the CAR comprises a PSMA binding domain comprising the amino acid sequence set forth in SEQ ID NO: 13; and

a dominant negative receptor comprising the amino acid sequence set forth in SEQ ID NO: 115.

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